Arcadi Gual

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.

Human myofibroblastic hepatic stellate cells express Ca2+-activated K+ channels that modulate the effects of endothelin-1 and nitric oxide

BACKGROUND/AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.

Facility changes mediated by cAMP in the bovine anterior segment in vitro.

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. Anterior segments were perfused in vitro at a constant pressure of 10 mmHg for 270 min with a general protocol as follows: 90 min control perfusion with DMEM, 90 min of experimental perfusion with DMEM containing the test drug(s), and 90 min of postdrug-perfusion with DMEM. C was calculated as the ratio between the rate of medium inflow (microliter/min) and the perfusion pressure (mmHg). Anterior segments can be perfused in vitro for up to 5 hr without significantly modifying their C. The addition of epinephrine, forskolin, dibutyryl-cAMP or isobutylmethylxanthine to the control perfusion medium elicited a significant increase of C. If, during isobutylmethylxanthine perfusion, forskolin or epinephrine was added, C increased significantly. Finally, perfusion with indomethacin prior to addition of epinephrine prevented the increase of C induced by epinephrine. Epinephrine, the adenylate cyclase activator forskolin, the cAMP analog dibutyryl-cAMP, and the phosphodiesterase inhibitor isobutylmethylxanthine all increase aqueous facility. It seems reasonable to suspect that the cAMP system is involved in epinephrines effects on bovine trabecular meshwork cells. Moreover, the complete inhibition by indomethacin of the outflow facility increase induced by epinephrine suggests that prostaglandins may be involved in the outflow facility mechanisms related to adrenoreceptor stimulation of trabecular meshwork cells.

Acid-sensing ion channels detect moderate acidifications to induce ocular pain.

Abstract Sensory nerve fibers innervating the ocular anterior surface detect external stimuli producing innocuous and painful sensations. Protons are among the first mediators released by damaged cells during inflammation, tissue injury, or other chronic ophthalmic conditions. We studied whether acid-sensing ion channels (ASICs) are expressed in corneal sensory neurons and their roles in the response to moderate acidifications of the ocular surface and in pathologies producing ocular surface inflammation. Moderate acidic pH (6.6) activated ASIC-like currents in corneal sensory neurons, which were blocked by ASIC1- or ASIC3-specific toxins. Acidic pH depolarizes corneal sensory neurons to fire action potentials, an effect blocked by the ASIC3 inhibitor APETx2. 2-Guanidino-4-methylquinazoline, an ASIC3 agonist, activated a population of corneal polymodal sensory nerve fibers and significantly increased the blinking and tearing rate. The nocifensive behaviors produced by application of either a moderate acidic stimulus or ophthalmic drugs formulated in acidic solution were abolished by ASIC blockers. In a model of allergic keratoconjunctivitis, nocifensive behavior was greatly reduced by ASIC3 blockade, presumably by reducing nociceptor sensitization during the inflammatory process. Our results show that, in addition to the established role of TRPV1, ASICs play a significant role in the detection of acidic insults at the ocular surface. The identification of ASICs in corneal neurons and their alterations during different diseases is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies.

Glaucoma patients present increased levels of diadenosine tetraphosphate, Ap4A, in the aqueous humour

Previous studies have shown the presence of diadenosine tetraphosphate (Ap(4)A) and pentaphosphate (Ap(5)A) in the aqueous humour (AH) of different species. When topically applied to the rabbit cornea, Ap(4)A decreased IOP while Ap(5)A increased it. Here we study the presence of dinucleoside polyphosphates in the AH from human patients with or without glaucoma. AH was obtained at the time of cataract surgery from patients with (n=16) or without (n=10) primary open-angle glaucoma. AH (0.1-0.2 ml) was collected at the beginning of surgery through a corneal paracentesis and immediately cooled in liquid nitrogen, kept frozen and protected from light. AH aliquots were analyzed by HPLC for the presence of Ap(4)A and Ap(5)A. Both, Ap(4)A and Ap(5)A were detected in the AH of both experimental groups. No significant differences were found for Ap(5)A. In contrast, Ap(4)A levels were increased by ∼15-fold in the AH from glaucomatous eyes ranging from 19.5±9.2 nM in normal individuals to 286.03±30.9 nM in glaucomatous patients. In conclusion, both Ap(4)A and Ap(5)A were detected for the first time in human AH. Interestingly, glaucomatous eyes presented elevated concentrations of Ap(4)A compared to controls. The role of Ap(4)A needs to be elucidated but it may help to protect the autonomic innervation in the ciliary body/trabecular meshwork. Also, because of its higher levels in glaucoma patients it may be considered as a possible glaucoma biomarker.

Defining the learning outcomes of graduates from the medical school at the University of Barcelona (Catalonia, Spain).

It is generally accepted that medical schools must clearly define learning outcomes for their students. During the process of curriculum change initiated in 1990, Spanish medical schools introduced a range of general objectives but no specific outcomes were defined. In 2001, in an effort to improve its curriculum, the Medical School at the University of Barcelona decided to define the specific learning outcomes for its graduates. The process was carried out by a teachers’ group, comprising individuals from different branches of medicine, drawing largely on the Outcome-based Education in Medicine model introduced by the Scottish Deans’ Medical Curriculum Group (2000). Other different stakeholders were asked to give any suggestions for modifications in order to prepare a definitive document to be approved by the medical school. The whole process took two years to complete. The authors discuss the advantages of such a process for students, teachers and the institution.

Profilin induces lamellipodia by growth factor-independent mechanism

Profilin has been implicated in cell mo tility 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 lamellipo‐ dia. 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 lamel‐ lipodia 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)

Mathematical analysis of epidural space location.

BackgroundThe location of epidural space for local anaesthetic injection can be difficult. The aim of this study was to define the mathematical function of the pressure changes in the syringe during puncture of the epidural space. Knowledge of pressure changes might be of help to the anesthetist who attempts to ascertain the location of the needle, and it is essential to the design of a device with which to locate epidural space.MethodsEpidural punctures were performed in 20 patients, using an 18-Tuohy needle connected to a 10 ml syringe. The epidural space was located by theloss of resistance technique. Pressure variations within the injection system during epidural puncture were measured and digitized at 250 Hz. Pressure curves were analyzed for amplitude and rate of a decay after entry of the needle into the epidural space.ResultsPressure increased as the needle passed through skin, subcutaneous fat and muscle. The maximal pressure was observed when the needle perforated theligamentum flavum (689±124 cm H2O). When the needle entered the epidural space, an exponential decrease in pressure was observed in all patients (R2=0.99; τ=2.1±0.9 seconds). End-residual pressure was 22±12 cm H2O. The change in pressure observed when the needle entered the epidural space fitted a negative exponential function (y=e−x/2.08).ConclusionsPressures within the injection system for epidural puncture can reach 1100 cm H2O. Location of the epidural space is characterized by an exponential decay to and end-residual pressure below 50 cm H2O, with a constant time of approximately 2 seconds.

Medical education in Spain: current status and new challenges

As in other countries, medical education in Spain is structured across three distinct stages: undergraduate or basic medical education; postgraduate specialized training; and continuing medical education. The aim of this article is to give an overview of the current state of these three stages, discussing the strengths and weaknesses and the challenges facing each one in the coming years, and how Spain can look to the international community to support change. We suggest that the undergraduate medical education system should be adapted to Spains new social requirements and requires to be increasingly aligned with postgraduate training. We suggest that continuing medical education should develop its Continuous Professional Development programmes to ensure the permanent competence of Spanish medical professionals. The European Higher Education arena, as defined by the Bologna Declaration, provides many opportunities as well as a challenging situation for improving any current weaknesses in the Spanish medical education system.

Activation of Store-Operated Ca 2 Channels in Trabecular Meshwork Cells

PURPOSE In nonexcitable cells, G(q)-coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca(2+)](i)) expressed as an initial IP(3)-dependent release from intracellular stores followed by a sustained Ca(2+) influx from the extracellular space that involves store-operated Ca(2+) channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca(2+) signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility. METHODS [Ca(2+)](i) was measured in cultured bovine TM cells loaded with Fura-2. Ca(2+) currents were recorded using the patch clamp technique. Cell contractility measurements were assessed by traction microscopy. RESULTS BK and ET-1 activate a store-operated Ca(2+) entry that was greatly reduced in the absence of extracellular Ca(2+) or by preincubation with SOC blocker 2-APB or SKF96365. Store-operated Ca(2+) currents were also activated by intracellular dialysis with IP(3) + EGTA or after stimulation with thapsigargin. Electrophysiological characterization supports the presence of Ca(2+) release-activated Ca(2+) channels (CRACs) and nonselective cation channels, of which TRPC1 and TRPC4 channels may be candidate TRPs detected in TM cells. Extracellular Ca(2+) entry through SOCs is not required for TM cell contraction in response to BK or ET-1, but it modulates this process. CONCLUSIONS Extracellular Ca(2+) entry in TM cells in response to agonist stimulation and store-depletion is mediated by the activation of SOCs, which do not contribute to cell contraction but which may activate regulatory mechanisms to prevent excessive contraction. CRAC and TRPC channels involved represent interesting modulators of TM function to improve aqueous humor outflow.