Resumen Objetivo: Estudiar la presencia de polifosfatos de diadenosina en el humor acuoso de huma... more Resumen Objetivo: Estudiar la presencia de polifosfatos de diadenosina en el humor acuoso de humanos. Objetivo secundario: Analizar si existen diferencias entre pacientes con y sin glaucoma. Método: Recogida de muestras de humor acuoso de 20 ojos de 20 pacientes subsidiarios de cirugía de cataratas. La extracción se realizó en la primera maniobra quirúrgica de la facoemulsificación: a través de córnea clara se realizó una paracentesis y se aspiraron de 0,1 a 0,2 ml de humor acuoso con una cánula de 27 G conectada a una jeringa de insulina. La muestra se congeló inmediatamente a -80 ºC y se mantuvo protegida de la luz hasta que se analizó la presencia de AP 4 A y Ap 5 A por cromatografía líquida de alta resolución. Resultados: Diez pacientes presentaban glaucoma (9 glaucoma primario de ángulo abierto y uno glaucoma seudoexfoliativo). Se detectaron AP 4 A y AP 5 A en todos los pacientes. No se detectaron diferencias en la concentración de AP 5 A entre los pacientes con y sin glaucoma. Las concentraciones de AP 4 A fueron más elevadas que las del AP 5 A en el grupo con glaucoma y se detectó una diferencia estadísticamente significativa con el grupo control (296,5 nM en el grupo glaucoma frente a 27,5 nM en el grupo control). Conclusiones: Por primera vez se detecta la presencia de AP 4 A y AP 5 A en humor acuoso humano. En los pacientes con glaucoma las concentraciones de AP 4 A están elevadas en comparación a los controles. Aunque se ha descrito el papel regulador de la presión intraocular del AP 4 A en modelos animales la importancia fisiopatológica de estas moléculas en humanos todavía no se conoce.
To study the presence of inwardly rectifying K ϩ (Kir) channels in cultured bovine (BTM) and huma... more To study the presence of inwardly rectifying K ϩ (Kir) channels in cultured bovine (BTM) and human (HTM) trabecular meshwork cells. METHODS. Cultures of BTM and HTM cells were obtained by an extracellular matrix digestion technique. Whole-cell patchclamp recordings of BTM cells were performed with the appropriate solutions to detect K ϩ currents. Also, Western blot analysis of Kir2.1 protein expression was performed on both cultured BTM and HTM cells.
Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through ... more Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α 1 adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigate the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory and fibrogenic actions in the injured liver. Methods: Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular calcium concentration ([Ca 2+ ] i ) was studied in fura-2 loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC-II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by 3 H-thymidine incorporation and quantitative PCR, respectively. NFκB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Results: Normal human livers expressed α 1A adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α 1A adrenoceptors. Norepinephrine induced multiple rapid [Ca 2+ ] i oscillations (Ca 2+ spikes). Prazosin (α 1 blocker) completely prevented NE-induced Ca 2+ spikes, while propranolol (nonspecific β blocker) partially attenuated this effect. Norepinephrine caused phosphorylation of MLC-II, and cell contraction. In constrast, NE did not affect cell proliferation nor collagen α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. Norepinephrine stimulated NFκB activation. BAY11-7082, a specific NFκB inhibitor, blocked NE-induced chemokine secretion.
In the eye, trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor... more In the eye, trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow. Among their functions, ClC-2 chloride channels are thought to be involved in regulation of cellular volume and intracellular [Cl−]. We characterized the properties and modulation of an inwardly rectifying chloride current activated in these cells. Patch-clamp recordings revealed inwardly rectifying chloride currents activated
Abstracts of the 3rd ECCO Congress, Lyon, France, February 28-March 1, 2008 81 newly diagnosed an... more Abstracts of the 3rd ECCO Congress, Lyon, France, February 28-March 1, 2008 81 newly diagnosed and untreated. Serum concentrations of free soluble RANKL, OPG, TNF-α, IL-1β, IL-6, osteocalcin and C-telopeptide type I were measured by immunoassay. Reference ranges were derived from 30 age-matched healthy controls. Bone mineral density (BMD) of the spine and total hip was measured by DXA. Results: We found 53% of Crohn' s patients with reduced BMD (t-score≤1.0) at diagnosis, and low bone mass in 72% of the study population. Elevated concentrations of sRANKL, OPG, TNF-α and IL-6 were found in patients with bone disease (p<0.01). In the newly diagnosed, previously untreated patients there was a good association between TNF-α and free sRANKL (r=0.6; p=0.027), and this positive correlation remained unchanged in the unselected study population as a whole (r=0.5; p=0.002). Stepwise multiple regression indicated TNF-α to be the best predictor of sRANKL (p<0.001). Patients with increased parameter of bone resorption were characterized by elevation of TNF-α, IL-6, CRP and OPG in systemic circulation. Analysis of the OPG and sRANKL relationship according to subgroups showed absence of correlation in patients with healthy skeleton, and an inverse relationship in those with pathologic BMD (r=-0.36; p=0.003). In the newly diagnosed patients with reduced BMD, correlation between free sRANKL and OPG was highly inverse (r=-0.8; p=0.02), whereas in those with normal BMD there was no relationship. Conclusion: We found low bone mass in 50% of newly diagnosed and untreated Crohn' s patients. In naïve Crohn' s patients we demonstrated strong relationship between TNF-α and the osteoclastic mediator sRANKL, and this positive correlation persisted across the unselected study population. Free sRANKL and OPG showed highly inverse relationship in patients with pathological bone density but not in those with healthy skeleton. Data on the newly diagnosed patients support the prominent role of inflammation in bone loss. Therefore, bone disease that accompanies Crohn' s disease needs to be considered for therapeutic options already at the diagnosis.
Inflammation is a complex process that implies the interaction between cells and molecular mediat... more Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly "tuned, " can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P 1 ,P 4 -diadenosine tetraphosphate (Ap 4 A), and P 1 ,P 5 -diadenosine pentaphosphate (Ap 5 A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A 2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A 3 receptor, selective agonists like N 6 -(3-iodobenzyl)-5 -N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.
Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through ... more Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α 1 adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigate the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory and fibrogenic actions in the injured liver. Methods: Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular calcium concentration ([Ca 2+ ] i ) was studied in fura-2 loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC-II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by 3 H-thymidine incorporation and quantitative PCR, respectively. NFκB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Results: Normal human livers expressed α 1A adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α 1A adrenoceptors. Norepinephrine induced multiple rapid [Ca 2+ ] i oscillations (Ca 2+ spikes). Prazosin (α 1 blocker) completely prevented NE-induced Ca 2+ spikes, while propranolol (nonspecific β blocker) partially attenuated this effect. Norepinephrine caused phosphorylation of MLC-II, and cell contraction. In constrast, NE did not affect cell proliferation nor collagen α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. Norepinephrine stimulated NFκB activation. BAY11-7082, a specific NFκB inhibitor, blocked NE-induced chemokine secretion.
The aim of this study was to investigate the influence of substances that increase intracellular ... more The aim of this study was to investigate the influence of substances that increase intracellular cAMP levels on the aqueous humor outflow facility (C) of isolated bovine anterior segments.
Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health ... more Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health concern. The etiology and pathophysiology of postoperative pain are still poorly understood, but hydrogen ions appear to be important in this process. We have investigated the role of peripheral acid-sensing ion channels (ASICs), which form depolarizing channels activated by extracellular protons, in a rat model of postoperative pain (i.e., hindpaw skin/muscle incision). We report high levels of ASIC-type currents (ϳ77%) in sensory neurons innervating the hindpaw muscles, with a prevalence of ASIC3-like currents. The ASIC3 protein is largely expressed in lumbar DRG neurons innervating the plantar muscle, and its mRNA and protein levels are increased by plantar incision 24 h after surgery. Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone. The specific homomeric ASIC1a blocker PcTx1 has no effect on spontaneous flinching, when applied peripherally. Together, these data demonstrate a significant role for peripheral ASIC3-containing channels in postoperative pain.
The possibility of changing the number of synapses may be an important asset in the treatment of ... more The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.
Profilin has been implicated in cell motility and in a variety of cellular processes, such as mem... more Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time-and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.-Syriani, E., Gomez-Cabrero, A., Bosch, M., Moya, A., Abad, E., Gual, A., Gasull, X., Morales, M. Profilin induces lamellipodia by growth factor independent mechanism. FASEB J. 22, 1581-1596 (2008)
Proceedings of the National Academy of Sciences, 2012
In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particula... more In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments. sodium channels | nociception A cid-sensing ion channels (ASICs) are depolarizing cationic channels gated by extracellular protons (1-3). Four genes encoding at least six subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4) have been identified so far in rodents. Functional channels have been proposed to result from the trimeric association of subunits (4), leading to homomeric or heteromeric channels. ASICs are largely expressed in neurons, both in central and peripheral nervous systems. Whereas ASIC1a and ASIC2 are widely present in the rodent nervous system, the expression of ASIC1b and ASIC3 is primarily restricted to sensory neurons (5-7). The ASIC3 subunit is highly expressed in rat nociceptive neurons (8, 9). The expression pattern of ASIC subunits is less well documented in humans, where ASIC3 (10-12) has three variants differing in their C-termini (2). The physiological relevance and properties of these human variants have so far never been studied.
a b s t r a c t a r t i c l e i n f o Tissue acidosis is a common feature of many painful conditi... more a b s t r a c t a r t i c l e i n f o Tissue acidosis is a common feature of many painful conditions. Protons are indeed among the first factors released by injured tissues, inducing a local pH fall that depolarizes peripheral free terminals of nociceptors and leads to pain. ASICs are excitatory cation channels directly gated by extracellular protons that are expressed in the nervous system. In sensory neurons, they act as "chemo-electrical" transducers and are involved in somatic and visceral nociception. Two highly specific inhibitory peptides isolated from animal venoms have considerably helped in the understanding of the physiological roles of these channels in pain. At the peripheral level, ASIC3 is important for inflammatory pain. Its expression and its activity are potentiated by several pain mediators present in the "inflammatory soup" that sensitize nociceptors. ASICs have also been involved in some aspects of mechanosensation and mechanonociception, notably in the gastrointestinal tract, but the underlying mechanisms remain to be determined. At the central level, ASIC1a is largely expressed in spinal cord neurons where it has been proposed to participate in the processing of noxious stimuli and in central sensitization. Blocking ASIC1a in the spinal cord also produces a potent analgesia in a broad range of pain conditions through activation of the opiate system. Targeting ASIC channels at different levels of the nervous system could therefore be an interesting strategy for the relief of pain.
Background/Aims: Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis ... more Background/Aims: Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis and portal hypertension. Knowledge on human HSCs is based on studies using HSCs isolated from normal livers. We investigated the phenotypic, genomic and functional characteristics of HSCs from human cirrhotic livers.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015
In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic ... more In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic equilibrium between positive and negative regulatory factors. We hypothesized that neuronal pentraxin (NP1), a proapoptotic protein induced by low neuronal activity, could be a negative regulator of synapse density because it is found in dystrophic neurites in Alzheimer's disease-affected brains. Here, we report that knockdown of NP1 increases the number of excitatory synapses and neuronal excitability in cultured rat cortical neurons and enhances excitatory drive and long-term potentiation in the hippocampus of behaving mice. Moreover, we found that NP1 regulates the surface expression of the Kv7.2 subunit of the Kv7 family of potassium channels that control neuronal excitability. Furthermore, pharmacological activation of Kv7 channels prevents, whereas inhibition mimics, the increase in synaptic proteins evoked by the knockdown of NP1. These results indicate that NP1 negatively reg...
High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive facto... more High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK(Ca) channels and what their role in the regulation of HSCs contractility is. The presence of BK(Ca) channels in HSCs was assessed by the patch-clamp technique. The functional role of BK(Ca) channels was investigated by measuring intracellular calcium concentration ([Ca(2+)](i)) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK(Ca) channels. BK(Ca) channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK(Ca) channel blocker, increased both the sustained phase of [Ca(2+)](i) elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK(Ca) channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK(Ca) channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca(2+)](i) increase and cell contraction. Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.
It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractili... more It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractility in the trabecular meshwork (TM). Several substances known to contract the TM reduce outflow facility, whereas cellular relaxation is commonly associated with the opposite effect. Inhibitors of the RhoA pathway are under development as antiglaucoma drugs. Here the authors investigate the role of platelet-derived growth factor (PDGF), a known activator of the Rac1 pathway, in cell cytoskeleton, outflow facility, and intraocular pressure (IOP). METHODS. Effects of PDGF on actin cytoskeleton, Rac1, and AKT activation were tested in preconfluent and confluent bovine TM cells in culture. Rac1 and AKT/P-AKT activation were assessed by Western blot analysis. Trabecular outflow facility was measured in bovine perfused anterior segments. Changes in IOP were measured for up to 6 hours after topical application in the cornea of rabbit eyes by means of a contact tonometer. RESULTS. In TM cells, PDGF (10 ng/mL) activated Rac1 through AKT and induced actin cytoskeleton rearrangement with lamellipodia formation. In this sense, lamellipodia formation in TM cells was prevented by NSC23766, a Rac1 inhibitor, and LY294002, a PI3K inhibitor. In perfused anterior segments, PDGF (100 ng/mL) increased trabecular outflow facility by 26%. In vivo, when topically applied to rabbit corneas, PDGF induced a 20% decrease in IOP (100 ng/mL). This reduction was concentration dependent and presented an EC 50 value of 2.7 nM. CONCLUSIONS. PDGF, by activating the Rac1 pathway, induces cytoskeletal changes in TM cells that enhance outflow facility. Decreased IOP after PDGF application is likely caused by the facilitation of aqueous humor outflow. Rac1 pathway activation appears to be a positive modulator of outflow facility and an interesting target for decreasing IOP after ocular hypertension. (Invest Ophthalmol Vis Sci.
In nonexcitable cells, G q -coupled membrane receptor activation induces a biphasic increase in i... more In nonexcitable cells, G q -coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca 2ϩ ] i ) expressed as an initial IP 3 -dependent release from intracellular stores followed by a sustained Ca 2ϩ influx from the extracellular space that involves store-operated Ca 2ϩ channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca 2ϩ signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility.
PURPOSE. Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with e... more PURPOSE. Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with extracellular matrix determine outflow facility. Because cell volume seems essential to TM function, this study was conducted to investigate further the ionic channels and receptors involved in regulatory volume decrease and their roles in modulating outflow facility. METHODS. Primary cultures of bovine TM cells were used. K ϩ and Cl Ϫ currents were studied with whole-cell patch clamping. Swelling was induced by hypotonic shock. [Ca 2ϩ ] i was measured in TM cells loaded with fura-2. Bovine anterior segments were perfused at constant pressure to measure outflow facility. RESULTS. Hypotonic media activated both the high-conductance Ca 2ϩ -activated K ϩ channel (BK Ca ) and swelling-activated Cl Ϫ channel (Cl swell ) currents and induced release of adenosine 5Ј-triphosphate (ATP) from TM cells. ATP activated P2Y 2 receptors with the following profile: ATP ϭ uridine 5Ј-triphosphate (UTP) Ͼ adenosine 5Ј-O-(3-thiotriphosphate) (ATP-␥ S) Ͼ adenosine 5Ј-diphosphate (ADP) ϭ uridine 5Ј-diphosphate (UDP), and increased BK Ca current. Hypotonic medium initially decreased outflow facility in perfused anterior segments, which recovered with time to baseline levels. Addition of tamoxifen or iberiotoxin (Cl swell and BK Ca blockers, respectively) lengthened the recovery phase, which implies that these channels participate in cell volume regulation. In contrast, an activator of BK Ca s (NS1619) produced the opposite effect. CONCLUSIONS. Cell swelling activates a regulatory volume decrease mechanism that implies activation of K ϩ and Cl Ϫ currents and participation of P2Y 2 receptors. Because previous studies have shown that intracellular volume of TM cells is an important determinant of outflow facility, it seems feasible that cell volume regulation would be part of the homeostatic mechanisms of the TM, to regulate the outflow pathway. (Invest Ophthalmol Vis Sci.
Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibrobla... more Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblast-like cells, which develop contractile properties and contribute to increased resistance to blood flow. We investigated whether this phenotypic activation includes changes in the expression of L-type voltage-operated Ca 2؉ channels (VOCC), which mediate Ca 2؉ influx and regulate cell contraction in vascular cell types. Rat HSCs were studied in the quiescent phenotype and after their activation in vitro (cultured on plastic for 14 days) and in vivo (isolated from rats with CCl 4 -induced cirrhosis). Patch-clamp studies showed Ca 2؉ currents through L-type VOCC in HSCs activated both in vitro and in vivo, whereas no currents were detected in quiescent HSCs. Moreover, binding studies with 3 H-isradipine, a specific L-type VOCC antagonist, showed a large number of binding sites in activated HSCs, while no specific binding was found in quiescent HSCs. Finally, messenger RNA (mRNA) encoding L-type VOCC was not detected in quiescent HSCs as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis, whereas it was present in activated HSCs. Stimulation of L-type VOCC with KCl resulted in a marked increase in [Ca 2؉ ] i followed by cell contraction in HSCs activated both in vitro and in vivo, whereas no effects were observed in quiescent HSCs. We conclude that the activation of HSCs is associated with up-regulation of L-type VOCC that mediate Ca 2؉ influx and cell contraction. These results may be relevant to the pathogenesis of portal hypertension.
Resumen Objetivo: Estudiar la presencia de polifosfatos de diadenosina en el humor acuoso de huma... more Resumen Objetivo: Estudiar la presencia de polifosfatos de diadenosina en el humor acuoso de humanos. Objetivo secundario: Analizar si existen diferencias entre pacientes con y sin glaucoma. Método: Recogida de muestras de humor acuoso de 20 ojos de 20 pacientes subsidiarios de cirugía de cataratas. La extracción se realizó en la primera maniobra quirúrgica de la facoemulsificación: a través de córnea clara se realizó una paracentesis y se aspiraron de 0,1 a 0,2 ml de humor acuoso con una cánula de 27 G conectada a una jeringa de insulina. La muestra se congeló inmediatamente a -80 ºC y se mantuvo protegida de la luz hasta que se analizó la presencia de AP 4 A y Ap 5 A por cromatografía líquida de alta resolución. Resultados: Diez pacientes presentaban glaucoma (9 glaucoma primario de ángulo abierto y uno glaucoma seudoexfoliativo). Se detectaron AP 4 A y AP 5 A en todos los pacientes. No se detectaron diferencias en la concentración de AP 5 A entre los pacientes con y sin glaucoma. Las concentraciones de AP 4 A fueron más elevadas que las del AP 5 A en el grupo con glaucoma y se detectó una diferencia estadísticamente significativa con el grupo control (296,5 nM en el grupo glaucoma frente a 27,5 nM en el grupo control). Conclusiones: Por primera vez se detecta la presencia de AP 4 A y AP 5 A en humor acuoso humano. En los pacientes con glaucoma las concentraciones de AP 4 A están elevadas en comparación a los controles. Aunque se ha descrito el papel regulador de la presión intraocular del AP 4 A en modelos animales la importancia fisiopatológica de estas moléculas en humanos todavía no se conoce.
To study the presence of inwardly rectifying K ϩ (Kir) channels in cultured bovine (BTM) and huma... more To study the presence of inwardly rectifying K ϩ (Kir) channels in cultured bovine (BTM) and human (HTM) trabecular meshwork cells. METHODS. Cultures of BTM and HTM cells were obtained by an extracellular matrix digestion technique. Whole-cell patchclamp recordings of BTM cells were performed with the appropriate solutions to detect K ϩ currents. Also, Western blot analysis of Kir2.1 protein expression was performed on both cultured BTM and HTM cells.
Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through ... more Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α 1 adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigate the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory and fibrogenic actions in the injured liver. Methods: Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular calcium concentration ([Ca 2+ ] i ) was studied in fura-2 loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC-II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by 3 H-thymidine incorporation and quantitative PCR, respectively. NFκB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Results: Normal human livers expressed α 1A adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α 1A adrenoceptors. Norepinephrine induced multiple rapid [Ca 2+ ] i oscillations (Ca 2+ spikes). Prazosin (α 1 blocker) completely prevented NE-induced Ca 2+ spikes, while propranolol (nonspecific β blocker) partially attenuated this effect. Norepinephrine caused phosphorylation of MLC-II, and cell contraction. In constrast, NE did not affect cell proliferation nor collagen α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. Norepinephrine stimulated NFκB activation. BAY11-7082, a specific NFκB inhibitor, blocked NE-induced chemokine secretion.
In the eye, trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor... more In the eye, trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow. Among their functions, ClC-2 chloride channels are thought to be involved in regulation of cellular volume and intracellular [Cl−]. We characterized the properties and modulation of an inwardly rectifying chloride current activated in these cells. Patch-clamp recordings revealed inwardly rectifying chloride currents activated
Abstracts of the 3rd ECCO Congress, Lyon, France, February 28-March 1, 2008 81 newly diagnosed an... more Abstracts of the 3rd ECCO Congress, Lyon, France, February 28-March 1, 2008 81 newly diagnosed and untreated. Serum concentrations of free soluble RANKL, OPG, TNF-α, IL-1β, IL-6, osteocalcin and C-telopeptide type I were measured by immunoassay. Reference ranges were derived from 30 age-matched healthy controls. Bone mineral density (BMD) of the spine and total hip was measured by DXA. Results: We found 53% of Crohn' s patients with reduced BMD (t-score≤1.0) at diagnosis, and low bone mass in 72% of the study population. Elevated concentrations of sRANKL, OPG, TNF-α and IL-6 were found in patients with bone disease (p<0.01). In the newly diagnosed, previously untreated patients there was a good association between TNF-α and free sRANKL (r=0.6; p=0.027), and this positive correlation remained unchanged in the unselected study population as a whole (r=0.5; p=0.002). Stepwise multiple regression indicated TNF-α to be the best predictor of sRANKL (p<0.001). Patients with increased parameter of bone resorption were characterized by elevation of TNF-α, IL-6, CRP and OPG in systemic circulation. Analysis of the OPG and sRANKL relationship according to subgroups showed absence of correlation in patients with healthy skeleton, and an inverse relationship in those with pathologic BMD (r=-0.36; p=0.003). In the newly diagnosed patients with reduced BMD, correlation between free sRANKL and OPG was highly inverse (r=-0.8; p=0.02), whereas in those with normal BMD there was no relationship. Conclusion: We found low bone mass in 50% of newly diagnosed and untreated Crohn' s patients. In naïve Crohn' s patients we demonstrated strong relationship between TNF-α and the osteoclastic mediator sRANKL, and this positive correlation persisted across the unselected study population. Free sRANKL and OPG showed highly inverse relationship in patients with pathological bone density but not in those with healthy skeleton. Data on the newly diagnosed patients support the prominent role of inflammation in bone loss. Therefore, bone disease that accompanies Crohn' s disease needs to be considered for therapeutic options already at the diagnosis.
Inflammation is a complex process that implies the interaction between cells and molecular mediat... more Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly "tuned, " can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P 1 ,P 4 -diadenosine tetraphosphate (Ap 4 A), and P 1 ,P 5 -diadenosine pentaphosphate (Ap 5 A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A 2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A 3 receptor, selective agonists like N 6 -(3-iodobenzyl)-5 -N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.
Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through ... more Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α 1 adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigate the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory and fibrogenic actions in the injured liver. Methods: Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular calcium concentration ([Ca 2+ ] i ) was studied in fura-2 loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC-II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by 3 H-thymidine incorporation and quantitative PCR, respectively. NFκB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Results: Normal human livers expressed α 1A adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α 1A adrenoceptors. Norepinephrine induced multiple rapid [Ca 2+ ] i oscillations (Ca 2+ spikes). Prazosin (α 1 blocker) completely prevented NE-induced Ca 2+ spikes, while propranolol (nonspecific β blocker) partially attenuated this effect. Norepinephrine caused phosphorylation of MLC-II, and cell contraction. In constrast, NE did not affect cell proliferation nor collagen α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. Norepinephrine stimulated NFκB activation. BAY11-7082, a specific NFκB inhibitor, blocked NE-induced chemokine secretion.
The aim of this study was to investigate the influence of substances that increase intracellular ... more The aim of this study was to investigate the influence of substances that increase intracellular cAMP levels on the aqueous humor outflow facility (C) of isolated bovine anterior segments.
Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health ... more Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health concern. The etiology and pathophysiology of postoperative pain are still poorly understood, but hydrogen ions appear to be important in this process. We have investigated the role of peripheral acid-sensing ion channels (ASICs), which form depolarizing channels activated by extracellular protons, in a rat model of postoperative pain (i.e., hindpaw skin/muscle incision). We report high levels of ASIC-type currents (ϳ77%) in sensory neurons innervating the hindpaw muscles, with a prevalence of ASIC3-like currents. The ASIC3 protein is largely expressed in lumbar DRG neurons innervating the plantar muscle, and its mRNA and protein levels are increased by plantar incision 24 h after surgery. Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone. The specific homomeric ASIC1a blocker PcTx1 has no effect on spontaneous flinching, when applied peripherally. Together, these data demonstrate a significant role for peripheral ASIC3-containing channels in postoperative pain.
The possibility of changing the number of synapses may be an important asset in the treatment of ... more The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.
Profilin has been implicated in cell motility and in a variety of cellular processes, such as mem... more Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time-and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.-Syriani, E., Gomez-Cabrero, A., Bosch, M., Moya, A., Abad, E., Gual, A., Gasull, X., Morales, M. Profilin induces lamellipodia by growth factor independent mechanism. FASEB J. 22, 1581-1596 (2008)
Proceedings of the National Academy of Sciences, 2012
In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particula... more In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments. sodium channels | nociception A cid-sensing ion channels (ASICs) are depolarizing cationic channels gated by extracellular protons (1-3). Four genes encoding at least six subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4) have been identified so far in rodents. Functional channels have been proposed to result from the trimeric association of subunits (4), leading to homomeric or heteromeric channels. ASICs are largely expressed in neurons, both in central and peripheral nervous systems. Whereas ASIC1a and ASIC2 are widely present in the rodent nervous system, the expression of ASIC1b and ASIC3 is primarily restricted to sensory neurons (5-7). The ASIC3 subunit is highly expressed in rat nociceptive neurons (8, 9). The expression pattern of ASIC subunits is less well documented in humans, where ASIC3 (10-12) has three variants differing in their C-termini (2). The physiological relevance and properties of these human variants have so far never been studied.
a b s t r a c t a r t i c l e i n f o Tissue acidosis is a common feature of many painful conditi... more a b s t r a c t a r t i c l e i n f o Tissue acidosis is a common feature of many painful conditions. Protons are indeed among the first factors released by injured tissues, inducing a local pH fall that depolarizes peripheral free terminals of nociceptors and leads to pain. ASICs are excitatory cation channels directly gated by extracellular protons that are expressed in the nervous system. In sensory neurons, they act as "chemo-electrical" transducers and are involved in somatic and visceral nociception. Two highly specific inhibitory peptides isolated from animal venoms have considerably helped in the understanding of the physiological roles of these channels in pain. At the peripheral level, ASIC3 is important for inflammatory pain. Its expression and its activity are potentiated by several pain mediators present in the "inflammatory soup" that sensitize nociceptors. ASICs have also been involved in some aspects of mechanosensation and mechanonociception, notably in the gastrointestinal tract, but the underlying mechanisms remain to be determined. At the central level, ASIC1a is largely expressed in spinal cord neurons where it has been proposed to participate in the processing of noxious stimuli and in central sensitization. Blocking ASIC1a in the spinal cord also produces a potent analgesia in a broad range of pain conditions through activation of the opiate system. Targeting ASIC channels at different levels of the nervous system could therefore be an interesting strategy for the relief of pain.
Background/Aims: Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis ... more Background/Aims: Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis and portal hypertension. Knowledge on human HSCs is based on studies using HSCs isolated from normal livers. We investigated the phenotypic, genomic and functional characteristics of HSCs from human cirrhotic livers.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015
In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic ... more In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic equilibrium between positive and negative regulatory factors. We hypothesized that neuronal pentraxin (NP1), a proapoptotic protein induced by low neuronal activity, could be a negative regulator of synapse density because it is found in dystrophic neurites in Alzheimer's disease-affected brains. Here, we report that knockdown of NP1 increases the number of excitatory synapses and neuronal excitability in cultured rat cortical neurons and enhances excitatory drive and long-term potentiation in the hippocampus of behaving mice. Moreover, we found that NP1 regulates the surface expression of the Kv7.2 subunit of the Kv7 family of potassium channels that control neuronal excitability. Furthermore, pharmacological activation of Kv7 channels prevents, whereas inhibition mimics, the increase in synaptic proteins evoked by the knockdown of NP1. These results indicate that NP1 negatively reg...
High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive facto... more High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK(Ca) channels and what their role in the regulation of HSCs contractility is. The presence of BK(Ca) channels in HSCs was assessed by the patch-clamp technique. The functional role of BK(Ca) channels was investigated by measuring intracellular calcium concentration ([Ca(2+)](i)) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK(Ca) channels. BK(Ca) channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK(Ca) channel blocker, increased both the sustained phase of [Ca(2+)](i) elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK(Ca) channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK(Ca) channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca(2+)](i) increase and cell contraction. Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.
It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractili... more It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractility in the trabecular meshwork (TM). Several substances known to contract the TM reduce outflow facility, whereas cellular relaxation is commonly associated with the opposite effect. Inhibitors of the RhoA pathway are under development as antiglaucoma drugs. Here the authors investigate the role of platelet-derived growth factor (PDGF), a known activator of the Rac1 pathway, in cell cytoskeleton, outflow facility, and intraocular pressure (IOP). METHODS. Effects of PDGF on actin cytoskeleton, Rac1, and AKT activation were tested in preconfluent and confluent bovine TM cells in culture. Rac1 and AKT/P-AKT activation were assessed by Western blot analysis. Trabecular outflow facility was measured in bovine perfused anterior segments. Changes in IOP were measured for up to 6 hours after topical application in the cornea of rabbit eyes by means of a contact tonometer. RESULTS. In TM cells, PDGF (10 ng/mL) activated Rac1 through AKT and induced actin cytoskeleton rearrangement with lamellipodia formation. In this sense, lamellipodia formation in TM cells was prevented by NSC23766, a Rac1 inhibitor, and LY294002, a PI3K inhibitor. In perfused anterior segments, PDGF (100 ng/mL) increased trabecular outflow facility by 26%. In vivo, when topically applied to rabbit corneas, PDGF induced a 20% decrease in IOP (100 ng/mL). This reduction was concentration dependent and presented an EC 50 value of 2.7 nM. CONCLUSIONS. PDGF, by activating the Rac1 pathway, induces cytoskeletal changes in TM cells that enhance outflow facility. Decreased IOP after PDGF application is likely caused by the facilitation of aqueous humor outflow. Rac1 pathway activation appears to be a positive modulator of outflow facility and an interesting target for decreasing IOP after ocular hypertension. (Invest Ophthalmol Vis Sci.
In nonexcitable cells, G q -coupled membrane receptor activation induces a biphasic increase in i... more In nonexcitable cells, G q -coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca 2ϩ ] i ) expressed as an initial IP 3 -dependent release from intracellular stores followed by a sustained Ca 2ϩ influx from the extracellular space that involves store-operated Ca 2ϩ channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca 2ϩ signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility.
PURPOSE. Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with e... more PURPOSE. Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with extracellular matrix determine outflow facility. Because cell volume seems essential to TM function, this study was conducted to investigate further the ionic channels and receptors involved in regulatory volume decrease and their roles in modulating outflow facility. METHODS. Primary cultures of bovine TM cells were used. K ϩ and Cl Ϫ currents were studied with whole-cell patch clamping. Swelling was induced by hypotonic shock. [Ca 2ϩ ] i was measured in TM cells loaded with fura-2. Bovine anterior segments were perfused at constant pressure to measure outflow facility. RESULTS. Hypotonic media activated both the high-conductance Ca 2ϩ -activated K ϩ channel (BK Ca ) and swelling-activated Cl Ϫ channel (Cl swell ) currents and induced release of adenosine 5Ј-triphosphate (ATP) from TM cells. ATP activated P2Y 2 receptors with the following profile: ATP ϭ uridine 5Ј-triphosphate (UTP) Ͼ adenosine 5Ј-O-(3-thiotriphosphate) (ATP-␥ S) Ͼ adenosine 5Ј-diphosphate (ADP) ϭ uridine 5Ј-diphosphate (UDP), and increased BK Ca current. Hypotonic medium initially decreased outflow facility in perfused anterior segments, which recovered with time to baseline levels. Addition of tamoxifen or iberiotoxin (Cl swell and BK Ca blockers, respectively) lengthened the recovery phase, which implies that these channels participate in cell volume regulation. In contrast, an activator of BK Ca s (NS1619) produced the opposite effect. CONCLUSIONS. Cell swelling activates a regulatory volume decrease mechanism that implies activation of K ϩ and Cl Ϫ currents and participation of P2Y 2 receptors. Because previous studies have shown that intracellular volume of TM cells is an important determinant of outflow facility, it seems feasible that cell volume regulation would be part of the homeostatic mechanisms of the TM, to regulate the outflow pathway. (Invest Ophthalmol Vis Sci.
Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibrobla... more Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblast-like cells, which develop contractile properties and contribute to increased resistance to blood flow. We investigated whether this phenotypic activation includes changes in the expression of L-type voltage-operated Ca 2؉ channels (VOCC), which mediate Ca 2؉ influx and regulate cell contraction in vascular cell types. Rat HSCs were studied in the quiescent phenotype and after their activation in vitro (cultured on plastic for 14 days) and in vivo (isolated from rats with CCl 4 -induced cirrhosis). Patch-clamp studies showed Ca 2؉ currents through L-type VOCC in HSCs activated both in vitro and in vivo, whereas no currents were detected in quiescent HSCs. Moreover, binding studies with 3 H-isradipine, a specific L-type VOCC antagonist, showed a large number of binding sites in activated HSCs, while no specific binding was found in quiescent HSCs. Finally, messenger RNA (mRNA) encoding L-type VOCC was not detected in quiescent HSCs as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis, whereas it was present in activated HSCs. Stimulation of L-type VOCC with KCl resulted in a marked increase in [Ca 2؉ ] i followed by cell contraction in HSCs activated both in vitro and in vivo, whereas no effects were observed in quiescent HSCs. We conclude that the activation of HSCs is associated with up-regulation of L-type VOCC that mediate Ca 2؉ influx and cell contraction. These results may be relevant to the pathogenesis of portal hypertension.
Uploads
Papers by Xavier Gasull