Fátima Ferreirinha

Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation

Background: Chronic pain may involve connective tissue remodeling due to inflammatory mediators. Results: Histamine H1 receptor activation causes ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels. Conclusion: Responses of skin fibroblasts to histamine are amplified by autocrine ATP release and P2Y1 purinoceptor activation. Significance: Amplification of histamine-mediated Ca2+ mobilization and growth of human fibroblasts by purines may be a novel therapeutic target for painful fibrotic diseases. Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

ATP released via pannexin-1 hemichannels mediates bladder overactivity triggered by urothelial P2Y6 receptors

In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y6 receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y6 receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y6 antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, (10)Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence video-microscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y6 receptors in the rat urothelium. Data indicate that activation of P2Y6 receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

BackgroundChronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts.ResultsBradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor.ConclusionsBradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Role of ecto-NTPDases on UDP-sensitive P2Y6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

This study aimed at investigating the expression and function of uracil nucleotide‐sensitive receptors (P2Y2, P2Y4, and P2Y6) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration‐dependently increased [Ca2+]i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y6 receptors with MRS 2578 prevented [Ca2+]i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y2, P2Y4, and P2Y6 receptors. While the expression of P2Y6 receptors remained fairly constant (7∼21 days), P2Y2 and P2Y4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, ‐2, and ‐3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP‐sensitive P2Y6 receptors coupled to increases in [Ca2+]i. Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation. J. Cell. Physiol. 227: 2694–2709, 2012.

Spinocerebellar ataxias in 114 Brazilian families: clinical and molecular findings.

To the Editor: Dominant spinocerebellar ataxias (SCAs) are a group of neurodegenerative diseases that affect the cerebellum, brain stem, and spinocerebellar tracts. SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17 and dentatorubral pallidoluysian atrophy (DRPLA) are caused by CAG trinucleotide repeat expansions (1). SCA8 has a CTG expansion in the causative gene. SCA10 has been found to represent a large expansion of a pentanucleotide (ATTCT) repeat (2). The frequency of each SCA varies between regions and ethnic groups. Machado–Joseph disease (MJD/SCA3) is the most common SCA among populations with Portuguese and Azorean background and populations in Japan and Germany (3–10). SCA2 is common in Cuba, India, Korea, and Italy (11– 14). SCA10 has been only described in the New World (15–19). Our aim was to determine the frequency of the SCA1, SCA2, SCA3, SCA6, SCA7, SCA10, SCA17 and DRPLA among SCA families who have the same geographic origin, namely southern Brazil (Rio Grande do Sul). Clinical, epidemiological, and molecular features are also described herein. Cases were recruited based on the following inclusion criteria: (1) ataxia, often associated with other neurological signs; and (2) inheritance as an autosomal dominant trait. Neurological examination followed a standard examination already published (19). Presence and gradation of several neurological findings were evaluated as numeric ordinal variables. Peripheral blood was collected, and genomic DNA was isolated from peripheral blood leukocytes, using the salting-out technique, as previously described (20). Molecular analyses of the repeat loci were performed by molecular tests specific to each ataxia by PCR amplification. Southern blot was performed to SCA10 locus. Patients were stratified according to their molecular diagnosis. Clinical variables were then compared between these groups. Comparisons were performed using analysis of variance (ANOVA), when the variable was parametric, or the Kruskal–Wallis test (KW test), when the variable was nonparametric. A Bonferroni procedure was done, in order to correct for multiple testing. This study was approved by the ethics committee of the institution where it was conducted; informed consent forms were filled out by all patients. A total of 114 families with autosomal dominant trait were investigated during an 8-year period (up to 2004). Diagnosis, ethnicity, and main clinical features are presented in Tables 1 and 2. SCA3 was the most prevalent, comprising 84.2% of all SCAs. The other SCAs were far less common and represented from one to five pedigrees each. The number of clinically examined patients with the diverse molecular diagnoses was variable. Due to this, some statistical comparisons could not be done: SCA1 and SCA10 patients were excluded from comparisons. Neurological findings that showed statistical differences among groups were (1) nystagmus, less frequent in cases with SCA2 and SCA7 than in the general sample; (2) limb ataxia, more severe in SCA6 patients; and (3) pyramidal findings and optic atrophy, more common in SCA7 patients than in other groups (p , 0.0025, 0.0025, 0.041 and 0.0411, respectively; KW test). Most of the present families were of mixed ancestry but Portuguese surnames prevailed. In our region, the main Portuguese ancestry was Azorean in origin, dating from circa 1750 (7). The Amerindians are another important ancestry. The composition of mitochondrial Amerindian markers in urban Brazilian populations is high and varies between 22% and 54% (26). This finding shows the importance of this Brazilian ancestry. So far, SCA10 has only been described in American families (15–18, 27). The finding of two SCA10 kindreds is in line with the hypothesis that this mutation is somehow restricted to patients with an Amerindian ancestry. Although some of the present families have already been described (7, 18, 19, 28), they had never been compared. We made these comparisons

Molecular diagnosis of Huntington disease in Portugal: implications for genetic counselling and clinical practice.

Huntington disease (HD) is a neurodegenerative, autosomal dominant disorder of late-onset, caused by the expansion of a CAG repeat in the coding region of the gene. Ours is the reference laboratory for genetic testing in HD, in Portugal, since 1998; 90.1% of all 158 families known were identified for the first time, including patients with unusual presentation or without family history. A total of 338 genetic tests were performed: 234 for diagnosis, 96 for presymptomatic and four for prenatal testing (four were done for family studies). Most referring physicians were neurologists (90.6%); 82.8% of all clinical diagnosis were confirmed, while 83.1% of those sent for exclusion were in fact excluded. In presymptomatic testing, an excess of female subjects (59.4%) was again verified; 37.5% of the consultands were found to be carriers. None of the foetuses, in four prenatal tests, were mutation carriers. One juvenile case was inherited from her mother. Our patient population is very similar to others described so far, namely in terms of mean age at onset and (CAG)n distribution, except perhaps for a higher frequency of large normal (class 2) alleles (3.7%). We also identify cases posing particular problems for genetic counselling, such as, ‘homozygosity’ that can pose a serious ethical dilemma, carriers of large normal alleles, and ‘homoallelism’ for a normal gene, which will demand further procedures and may delay results in presymptomatic and prenatal testing.

P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71 ± 3 yr, n=18). We focused on the mechanisms related to intracellular [Ca2+]i oscillations and plasma membrane‐dynamics. ATP, and the P2X7 agonist BzATP (100 μM), increased [Ca2+]i in parallel to the formation of membrane pores permeable to TO‐PRO‐3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7‐induced zeiosis was Ca2+ independent, but involved phospholipase C, protein kinase C, and Rho‐kinase activation. BzATP (100 μM) progressively increased the expression of Runx‐2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho‐kinase‐dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7‐Rho‐kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.—Noronha‐Matos, J. B., Coimbra, J., Sá‐e‐Sousa, A., Rocha, R., Marinhas, J., Freitas, R., Guerra‐Gomes, S., Ferreirinha, F., Costa, M. A., Correia‐de‐Sá, P., P2X7‐induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow‐derived mesenchymal stem cells FASEB J. 28, 5208–5222 (2014). www.fasebj.org

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum.

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A(2A) receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A(2B) and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A(2B) receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A(2A) receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A(2A) and A₃) receptors by endogenous adenosine. The selective A(2B) receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1-10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A(2A) receptors. In contrast to 2-Cl-IB MECA, activation of A(2A) receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A(2A) receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A(2A) receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

Despite the abundant expression of the UDP‐sensitive P2Y6 receptor in urothelial cells and sub‐urothelial myofibroblasts its role in the control of bladder function is not well understood.

Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.