Altered purinergic P2X7 and A2B receptors signaling limits macrophage-mediated host defense in schistosomiasis.

BACKGROUND: The occurrence of co-infections during schistosomiasis, a neglected tropical disease, with other parasites have been reported suggesting an impaired host immune defense. Macrophage purinergic P2X7 receptor (P2X7R) play an important role against intracellular pathogens. Therefore, we investigated the P2X7R-mediated phagocytosis and killing capacity of Leishmania amazonensis by macrophages during schistosomiasis in vitro and in vivo. METHODS: Swiss and C57BL/6 (Wild type) and P2X7R-/- were randomized in two groups: control (uninfected) and Schistosoma mansoni-infected. Alternatively, control Swiss and S. mansoni-infected mice were also infected with L. amazonensis. RESULTS: The pre-treatment of macrophages with the P2X7R antagonist (A74003) or TGF-ß reduced the phagocytosis index, mimicking the phenotype of cells from S. mansoni-infected mice and P2X7R-/- mice. Apyrase also reduced the phagocytosis index corroborating the role of ATP to macrophage activation. Moreover, l-arginine-nitric oxide pathway was compromised, which could explain the reduced killing capacity in response to ATP in vitro and in vivo. We found an increased extracellular nucleotide (ATP, ADP and AMP) hydrolysis along with an increased frequency of F4/80+ CD39+ macrophages from the S. mansoni-infected group. Moreover, the content of adenosine in the cell supernatant was higher in the S. mansoni-infected group in relation to controls. Schistosomiasis also increased the expression of macrophage adenosine A2BR. In good accordance, both ADA and the selective A2BR antagonist restored the phagocytosis index of macrophages from S. mansoni-infected group. CONCLUSIONS: Altogether, the altered P2X7R and A2BR signaling limits the role of macrophages to host defense against L. amazonensis during schistosomiasis, potentially contributing to the pathophysiology and clinically relevant co-infections.

P2Y2-P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis.

Schistosomiasis is an intravascular infectious disease that impacts over 200 million people globally. In its chronic stage, it leads to mesenteric inflammation with significant involvement of monocytes/macrophages. Endothelial cells lining the vessel lumens play a crucial role, and mount of evidence links this disease to a downregulation of endoprotective cell signaling favoring a primed and proinflammatory endothelial cell phenotype and therefore the loss of immunovascular homeostasis. One hallmark of infectious and inflammatory conditions is the release of nucleotides into the extracellular milieu, which, in turn, act as innate messengers, activating purinergic receptors and triggering cell-to-cell communication. ATP influences the progression of various diseases through P2X and P2Y purinergic receptor subtypes. Among these receptors, P2Y2 (P2Y2R) and P2X7 (P2X7R) receptors stand out, known for their roles in inflammation. However, their specific role in schistosomiasis has remained largely unexplored. Therefore, we hypothesized that endothelial P2Y2R and P2X7R could contribute to monocyte adhesion to mesenteric endothelial cells in schistosomiasis. Using a preclinical murine model of schistosomiasis associated with endothelial dysfunction and age-matched control mice, we showed that endothelial P2Y2R and P2X7R activation increased monocyte adhesion to cultured primary endothelial cells in both groups. However, a distinct upregulation of endothelial P2Y2R-driven canonical Ca2+ signaling was observed in the infected group, amplifying adhesion. In the control group, the coactivation of endothelial P2Y2R and P2X7R did not alter the maximal monocyte adhesion induced by each receptor individually. However, in the infected group, this coactivation induced a distinct upregulation of P2Y2R-P2X7R-driven canonical signaling, IL-1ß release, and VCAM-1 expression, with underlying mechanisms involving inflammasome and NF-κB signaling. Therefore, current data suggest that schistosomiasis alters endothelial cell P2Y2R/P2X7R signaling during inflammation. These discoveries advance our understanding of schistosomiasis. This intricate interplay, driven by PAMP-triggered endothelial P2Y2R/P2X7R cross-talk, emerges as a potential key player in the mesenteric inflammation during schistosomiasis.

The blockage of downstream P2Y2 receptor signaling inhibits the prostate cancer cell adhesion to endothelial cells.

AIMS: Prostate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. MAIN METHODS: For adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. KEY FINDINGS: Endothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SIGNIFICANCE: Endothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.

Unveiling the Potential of Purinergic Signaling in Schistosomiasis Treatment.

Schistosomiasis is a neglected tropical disease. It is related to long-lasting granulomatous fibrosis and inflammation of target organs, and current sub-optimal pharmacological treatment creates global public health concerns. Intravascular worms and eggs release antigens and extracellular vesicles that target host endothelial cells, modulate the immune system, and stimulate the release of damageassociated molecular patterns (DAMPs). ATP, one of the most studied DAMPs, triggers a cascade of autocrine and paracrine actions through purinergic P2X and P2Y receptors, which are shaped by ectonucleotidases (CD39). Both P2 receptor families, and in particular P2Y1, P2Y2, P2Y12, and P2X7 receptors, have been attracting increasing interest in several inflammatory diseases and drug development. Current data obtained from the murine model unveiled a CD39-ADP-P2Y1/P2Y12 receptors signaling pathway linked to the liver and mesenteric exacerbations of schistosomal inflammation. Therefore, we proposed that members of this purinergic signaling could be putative pharmacological targets to reduce schistosomal morbidity.

P2Y2 Receptor Induces L. amazonensis Infection Control in a Mechanism Dependent on Caspase-1 Activation and IL-1ß Secretion.

Leishmaniasis is a neglected tropical disease caused by an intracellular parasite of the genus Leishmania. Damage-associated molecular patterns (DAMPs) such as UTP and ATP are released from infected cells and, once in the extracellular medium, activate P2 purinergic receptors. P2Y2 and P2X7 receptors cooperate to control Leishmania amazonensis infection. NLRP3 inflammasome activation and IL-1ß release resulting from P2X7 activation are important for outcomes of L. amazonensis infection. The cytokine IL-1ß is required for the control of intracellular parasites. In the present study, we investigated the involvement of the P2Y2 receptor in the activation of NLRP3 inflammasome elements (caspase-1 and 11) and IL-1ß secretion during L. amazonensis infection in peritoneal macrophages as well as in a murine model of cutaneous leishmaniasis. We found that 2-thio-UTP (a selective P2Y2 agonist) reduced parasite load in L. amazonensis-infected murine macrophages and in the footpads and lymph nodes of infected mice. The antiparasitic effects triggered by P2Y2 activation were not observed when cells were pretreated with a caspase-1 inhibitor (Z-YVAD-FMK) or in macrophages from caspase-1/11 knockout mice (CASP-1,11-/-). We also found that UTP treatment induced IL-1ß secretion in wild-type (WT) infected macrophages but not in cells from CASP-1,11-/- mice, suggesting that caspase-1 activation by UTP triggers IL-1ß secretion in L. amazonensis-infected macrophages. Infected cells pretreated with IL-1R antagonist did not show reduced parasitic load after UTP and ATP treatment. Our in vivo experiments also showed that intralesional UTP treatment reduced both parasite load (in the footpads and popliteal lymph nodes) and lesion size in wild-type (WT) and CASP-11-/- but not in CASP-1,11-/- mice. Taken together, our findings suggest that P2Y2R activation induces CASP-1 activation and IL-1ß secretion during L. amazonensis infection. IL-1ß/IL-1R signaling is crucial for P2Y2R-mediated protective immune response in an experimental model of cutaneous leishmaniasis.

The α1-adrenoceptor-mediated human hyperplastic prostate cells proliferation is impaired by EGF receptor inhibition.

Benign prostatic hyperplasia (BPH) is an aging-related and progressive disease linked to an up-regulation of α1-adrenoceptors. The participation of EGF receptors (EGFR) in the GPCRs' signalosome has been described but so far data about the contribution of these receptors to prostatic stromal hyperplasia are scanty. We isolated and cultured vimentin-positive prostate stromal cells obtained from BPH patients. According to intracellular Ca2+ measurements, cell proliferation and Western blotting assays, these cultured hyperplastic stromal cells express functional α1-adrenoceptors and EGFR, and proliferate in response to the α1-adrenoceptor agonist phenylephrine. Interestingly, in these cells the inhibition of EGFR signaling with GM6001, CRM197, AG1478 or PD98059 was associated with full blockage of α1-adrenoceptor-mediated cell proliferation, while cell treatment with each inhibitor alone did not alter basal cell growth. Moreover, the co-incubation of AG1478 (EGFR inhibitor) with α1A/α1D-adrenoceptor antagonists showed no additive inhibitory effect. These findings highlight a putative role of EGFR signaling to α1-adrenoceptor-mediated human prostate hyperplasia, suggesting that the inhibition of this transactivation cascade could be useful to reduce BPH progression.

Revisiting the Pharmacodynamic Uroselectivity of α 1-Adrenergic Receptor Antagonists.

α1-Adrenoceptor (AR) antagonists are widely used for the relief of urinary retention secondary to benign prostatic hyperplasia (BPH). While the five Food and Drug Administration-approved α 1-AR antagonists (terazosin, doxazosin, alfuzosin, tamsulosin, and silodosin) share similar efficacy, they differ in tolerability, with reports of ejaculatory dysfunction. The aim of the present work was to revisit their α 1-AR subtype selectivity as well as of LDT5 (1-(2-methoxyphenyl)-4-[2-(3,4-dimethoxyphenyl) ethyl]piperazine monohydrochloride), a compound previously described as a multitarget antagonist of α 1A-/α 1D-AR and 5-HT1A receptors, and to estimate their affinity for D2, D3, and 5-HT1A receptors, which are putatively involved in ejaculatory dysfunction. Competition binding assays were performed with native (D2, 5-HT1A) or transfected (human α 1A-, α 1B-, α 1Dt-AR, and D3) receptors for determination of the drug's affinities. Tamsulosin and silodosin have the highest affinities for α 1A-AR, but only silodosin is clearly a selective α 1A-AR antagonist, with K i ratios of 25.3 and 50.2 for the α 1D- and α 1B-AR, respectively. Tamsulosin, silodosin, and LDT5 (but not terazosin, doxazosin, and alfuzosin) have high affinity for the 5-HT1A receptor (K i around 5-10 nM), behaving as antagonists. We conclude that the uroselectivity of tamsulosin is not explained by its too-low selectivity for the α 1A- versus α 1B-AR, and that its affinity for D2 and D3 receptors is probably too low for explaining the ejaculatory dysfunction reported for this drug. Present data also support the design of "better-than-LDT5" new multitarget lead compounds with pharmacokinetic selectivity based on poor brain penetration and that could prevent hyperplastic cell proliferation and BPH progression. SIGNIFICANCE STATEMENT: The present work revisits the uroselectivity of the five Food and Drug Administration-approved α1 adrenoceptor antagonists for the treatment of benign prostatic hyperplasia (BPH). Contrary to what has been claimed by some, our results indicate that the uroselectivity of tamsulosin is probably not fully explained by its too-weak selectivity for the α1A versus α1B adrenoceptors. We also show that tamsulosin affinity for D3 and 5-HT1A receptors is probably too low for explaining the ejaculatory dysfunction reported for this drug. Based on our lead compound LDT5, present data support the search for a multitarget antagonist of α1A-α1D and 5-HT1A receptors with poor brain penetration as an alternative for BPH treatment.

The P2Y1 receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin.

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y1 receptor (P2Y1R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y1R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y1R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y1R protein expression. In addition, assays with the Ca2+ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca2+ modulation. P2Y1R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y1 receptor signaling.

Purinergic signaling in schistosomal infection.

Human schistosomiasis is a chronic inflammatory disease caused by blood fluke worms belonging to the genus Schistosoma. Health metrics indicate that the disease is related to an elevated number of years lost-to-disability and years lost-to-life. Schistosomiasis is an intravascular disease that is related to a Th1 and Th2 immune response polarization, and the degree of polarization affects the outcome of the disease. The purinergic system is composed of adenosine and nucleotides acting as key messenger molecules. Moreover, nucleotide-transforming enzymes and cell-surface purinergic receptors are obligatory partners of this purinergic signaling. In mammalian cells, purinergic signaling modulates innate immune responses and inflammation among other functions; conversely purinergic signaling may also be modulated by inflammatory mediators. Moreover, schistosomes also express some enzymes of the purinergic system, and it is possible that worms modulate host purinergic signaling. Current data obtained in murine models of schistosomiasis support the notion that the host purinergic system is altered by the disease. The dysfunction of adenosine receptors, metabotropic P2Y and ionotropic P2X7 receptors, and NTPDases likely contributes to disease morbidity.

Increased expression of NTPDases 2 and 3 in mesenteric endothelial cells during schistosomiasis favors leukocyte adhesion through P2Y1 receptors.

Schistosomiasis is caused by an intravascular parasite and linked to phenotypic changes in endothelial cells that favor inflammation. Endothelial cells express P2Y1 receptors (P2Y1R), and their activation by ADP favors leukocyte adhesion to the endothelial monolayer. We aimed to evaluate the influence of schistosomiasis upon endothelial purinergic signaling-mediated leukocyte adhesion. Mesenteric endothelial cells and mononuclear cells from control and Schistosoma mansoni-infected mice were used in co-culture. P2Y1R levels were similar in both groups. Basal leukocyte adhesion was higher in the infected than in the control group; leukocyte adhesion increased after treatment with the P2Y1R agonist 2-MeSATP in both groups, though it only marginally increased in the infected group. Pre-incubation with the selective P2Y1R antagonist MRS2179 (0.3µM) prevented the agonist effect. However, in the infected group it also reduced the basal leukocyte adhesion, suggesting endothelial cell pre-activation. The endothelial expressions of NTPDases 2 and 3 were significantly increased in the infected group, increasing extracellular ATP hydrolysis and ADP formation by endothelial cells. Therefore, mesenteric endothelial cells are primed by schistosomiasis to a pro-inflammatory phenotype characterized by an increased expression of NTPDases 2 and 3, favoring ADP accumulation and mononuclear cell adhesion, possibly contributing to mesenteric inflammation and schistosomiasis morbidity.

New Multi-target Antagonists of α1A-, α1D-Adrenoceptors and 5-HT1A Receptors Reduce Human Hyperplastic Prostate Cell Growth and the Increase of Intraurethral Pressure.

Benign prostatic hyperplasia (BPH) is characterized by stromal cell proliferation and contraction of the periurethral smooth muscle, causing lower urinary tract symptoms. Current BPH treatment, based on monotherapy with α1A-adrenoceptor antagonists, is helpful for many patients, but insufficient for others, and recent reports suggest that stimulation of α1D-adrenoceptors and 5-hydroxytryptamine (serotonin) (5-HT)1A receptors contributes to cell proliferation. In this study, we investigated the potential of three N-phenylpiperazine derivatives (LDT3, LDT5, and LDT8) as multi-target antagonists of BPH-associated receptors. The affinity and efficacy of LDTs were estimated in isometric contraction and competition-binding assays using tissues (prostate and aorta) and brain membrane samples enriched in specific on- or off-target receptors. LDTs' potency was estimated in intracellular Ca(2+) elevation assays using cells overexpressing human α1-adrenoceptor subtypes. The antiproliferative effect of LDTs on prostate cells from BPH patients was evaluated by viable cell counting and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays. We also determined LDTs' effects on rat intraurethral and arterial pressure. LDT3 and LDT5 are potent antagonists of α1A-, α1D-adrenoceptors, and 5-HT1A receptors (Ki values in the nanomolar range), and fully inhibited phenylephrine- and 5-HT-induced proliferation of BPH cells. In vivo, LDT3 and LDT5 fully blocked the increase of intraurethral pressure (IUP) induced by phenylephrine at doses (ED50 of 0.15 and 0.09 µg.kg(-1), respectively) without effect on basal mean blood pressure. LDT3 and LDT5 are multi-target antagonists of key receptors in BPH, and are capable of triggering both prostate muscle relaxation and human hyperplastic prostate cell growth inhibition in vitro. Thus, LDT3 and LDT5 represent potential new lead compounds for BPH treatment.

Macrophage P2X7 receptor function is reduced during schistosomiasis: putative role of TGF- ß1.

Schistosomiasis is a chronic inflammatory disease whose macrophages are involved in immunopathology modulation. Although P2X7 receptor signaling plays an important role in inflammatory responses mediated by macrophages, no reports have examined the role of P2X7 receptors in macrophage function during schistosomiasis. Thus, we evaluated P2X7 receptor function in peritoneal macrophages during schistosomiasis using an ATP-induced permeabilization assay and measurements of the intracellular Ca(2+) concentration. ATP treatment induced significantly less permeabilization in macrophages from S. mansoni-infected mice than in control cells from uninfected animals. Furthermore, P2X7-mediated increases in intracellular Ca(2+) levels were also reduced in macrophages from infected mice. TGF-ß1 levels were increased in the peritoneal cavity of infected animals, and pretreatment of control macrophages with TGF-ß1 reduced ATP-induced permeabilization, mimicking the effect of S. mansoni infection. Western blot and qRT-PCR data showed no difference in P2X7 protein and mRNA between uninfected, infected, and TGF-ß1-treated groups. However, immunofluorescence analysis revealed reduced cell surface localization of P2X7 receptors in macrophages from infected and TGF-ß1-treated mice compared to controls. Therefore, our data suggest that schistosomiasis reduces peritoneal macrophage P2X7 receptor signaling. This effect is likely due to the fact that infected mice have increased levels of TGF-ß1, which reduces P2X7 receptor cell surface expression.

Pharmacological characterization of N1-(2-methoxyphenyl)-N4-hexylpiperazine as a multi-target antagonist of α1A/α1D-adrenoceptors and 5-HT1A receptors that blocks prostate contraction and cell growth.

Benign prostatic hyperplasia (BPH) is a progressive disease related to the imbalance of cell growth and apoptosis, and it plays a key role in the development of lower urinary tract symptoms (LUTS). The main pharmacological treatment is based on α1A-adrenoceptor blockers, but in several cases monotherapy has failed. Recent studies of prostate pathophysiology have noted the role of α1D-adrenoceptors and 5-HT1A receptors in prostate cell proliferation in addition to the usual role of α1A-adrenoceptors in prostate contraction. N-phenylpiperazine is a scaffold structure that may confer drug affinity for these three receptors. Therefore, the present work aimed to investigate the pharmacological characteristics of N1-(2-methoxyphenyl)-N4-hexylpiperazine (LDT66). Using isometric contraction assays with rat prostate and aorta, LDT66 reduced phenylephrine-induced contractions and showed K B values of 3.4 and 2.2 nM for α1A- and α1D-adrenoceptors, respectively. According to the functional binding assays data, LDT66 showed a high affinity (nanomolar range) for the 5-HT1A receptors, behaving as an antagonist. LDT66 also showed a low affinity (micromolar range) for receptors unrelated to BPH such as α1B-adrenoceptors, α2A-adrenoceptors, muscarinic and 5-HT2A receptors, which is a desirable profile in order to prevent putative side effects. Accordingly, LDT66 (100 µg/kg) showed a marginal hypotensive effect. Using the DU-145 prostate cells, control experiments characterized the α1D-adrenoceptor- and 5-HT1A receptor-mediated cell growth by phenylephrine and 5-HT, respectively. LDT66 (50 nM) prevented both effects similarly. In conclusion, LDT66 is a high-affinity multi-target antagonist of relevant receptors for BPH, and it may be a new starting point for multi-target drug development to treat BPH and LUTS.

Endothelial P2X7 receptors' expression is reduced by schistosomiasis.

Endothelial cells control vascular tone, permeability and leukocyte transmigration and are modulated by pro-inflammatory mediators. Schistosomiasis is an intravascular disease associated with inflammation, therefore altering endothelial cells' phenotype. Purinergic P2X7 receptors (P2X7R) play an important role in inflammation; however, the impact of the disease upon endothelial P2X7R function or expression has not been explored. Using ethidium bromide uptake to investigate P2X7R function, we observed that the effects of ATP (3 mM) and the P2X7R agonist 3'-O-(4-benzoyl)-ATP (BzATP) were smaller in mesenteric endothelial cells from the Schistosoma mansoni-infected group than in the control group. In the control group, BzATP induced endothelial nitric oxide production, which was blocked by the P2X7R antagonists KN-62 and A740003. However, in the infected group, we observed a reduced effect of BzATP and no effect of both P2X7R antagonists, suggesting a downregulation of endothelial P2X7R in schistosomiasis. We observed similar results in both infected and P2X7R(-/-) groups, which were also comparable to data obtained with KN-62- or A740004-treated control cells. Data from Western blot and immunocytochemistry assays confirmed the reduced expression of P2X7R in the infected group. In conclusion, our data show a downregulation of P2X7R in schistosomiasis infection, which likely limits the infection-related endothelial damage.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction.

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50% higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50 percent higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.

Δ²,³-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases.

Looking at a new putative target for the large spectrum antiparasitic drug ivermectin, we recently showed that avermectin-derived drugs are active against promastigote and amastigote forms of Leishmania amazonensis at low micromolar concentrations. However, we then reported that at this concentration range ivermectin is also able to inhibit three important mammalian P-type ATPases so that unacceptable adverse effects could occur if this drug were used at such high doses therapeutically. The present work aimed to test the activity of ten ivermectin analogs on these rat ATPases in search of a compound with similar leishmanicidal activity but with no effect on the mammalian (host) ATPases at effective concentrations. We synthesized three new ivermectin analogs for testing on rat SERCA (1a and 1b), Na+, K+-ATPase (α1 and α2/α3 isoforms) and H+/K+-ATPase activity, along with seven analogs already characterized for their leishmanicidal activity. Our main finding is that one of the prepared derivatives, Δ²,³-ivermectin ethyl secoester 8, is equipotent to ivermectin 1 for the in vitro leishmanicidal effects but is nearly without effect on the rat ATPases, indicating that it could have a better therapeutic index in vivo and could serve as a candidate for hit-to-lead progression. This conclusion is further supported by the fact that compound 8 produced only 6% (vs 77% for ivermectin) inhibition of the human kidney enzyme at 5 µM, a concentration corresponding to the IC50 for the activity against L. amazonensis amastigotes.

Ivermectin is a nonselective inhibitor of mammalian P-type ATPases.

Ivermectin is a large spectrum antiparasitic drug that is very safe at the doses actually used. However, as it is being studied for new applications that would require higher doses, we should pay attention to its effects at high concentrations. As micromolar concentrations of ivermectin have been reported to inhibit the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), we decided to investigate its putative inhibitory effect on other two important P-type ATPases, namely the Na(+) , K(+)-ATPase and H(+)/K(+)-ATPase. We first extended the data on SERCA, using preparations from rat enriched in SERCA1a (extensor digitorum longus) and 1b (heart) isoforms. Secondly, we tested the effect of ivermectin in two preparations of rat Na(+), K(+)-ATPase in order to appreciate its putative selectivity towards the alpha(1) isoform (kidney) and the alpha(2)/alpha(3) isoforms (brain), and in an H(+)/K(+)-ATPase preparation from rat stomach. Ivermectin inhibited all these ATPases with similar IC(50) values (6-17 microM). With respect to the inhibition of the Na(+), K(+)-ATPase, ivermectin acts by a mechanism different from the classical cardiac glycosides, based on selectivity towards the isoforms, sensibility to the antagonistic effect of K(+) and to ionic conditions favoring different conformations of the enzyme. We conclude that ivermectin is a nonselective inhibitor of three important mammalian P-type ATPases, which is indicative of putative important adverse effects if this drug were used at high doses. As a consequence, we propose that novel analogs of ivermectin should be developed and tested both for their parasitic activity and in vitro effects on P-type ATPases.

Melatonin inhibits LPS-induced NO production in rat endothelial cells.

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappaB) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappaB translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mum) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappaB pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity.

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity of alpha(2/3)-Na,K-ATPase varied with age, and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum.