José Gil-Longo

Calcium binding proteins.

The role of Ca(2+) as a key and pivotal second messenger in cells depends largely on a wide number of heterogeneous so-called calcium binding proteins (CBP), which have the ability to bind this ion in specific domains. CBP contribute to the control of Ca(2+) concentration in the cytosol and participate in numerous cellular functions by acting as Ca(2+) transporters across cell membranes or as Ca(2+)-modulated sensors, i.e., decoding Ca(2+) signals. In this chapter we review the main Ca(2+)-modulated CBP, starting with those intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins and calcineurin. Then, we address intracellular CBP lacking the EF-hand domain: CBP within intracellular Ca(2+) stores (paying special attention to calreticulin and calsequestrin), annexins and proteins that contain a C2 domain, such as protein kinase C (PKC) or sinaptotagmin. Finally, extracellular CBP have been classified in six groups, according to their Ca(2+) binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) γ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca(2+)-dependent (C)-type lectin-like domains; (vi) Ca(2+)-binding pockets of family C G-protein-coupled receptors. For all proteins, we briefly review their structure, location and function and additionally their potential as pharmacological targets in several human diseases.

Evaluation of the effects of several zoanthamine-type alkaloids on the aggregation of human platelets.

Ten zoanthamine-type alkaloids from two marine zoanthids belonging to the Zoanthus genus (Zoanthus nymphaeus and Zoanthus sp.) along with one semisynthetic derivative were evaluated for their antiplatelet activities on human platelet aggregation induced by several stimulating agents. 11-Hydroxyzoanthamine (11) and a synthetic derivative of norzoanthamine (16) showed strong inhibition against thrombin-, collagen- and arachidonic acid-induced aggregation, zoanthenol (15) displayed a selective inhibitory activity induced by collagen, while zoanthaminone (10) behaved as a potent aggregant agent. These evaluations allowed us to deduce several structure-activity relationships and suggest some mechanisms of action for this type of compounds.

Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta.

UNLABELLED Gallic acid autoxidation was monitored by absorption spectroscopy and H(2)O(2) production; vascular effects related to the autoxidation process were studied on intact and rubbed aortic rings from WKY rats. Gallic acid autoxidation in an oxygenated physiological salt solution (37 degrees C, pH=7.4) mostly occurred in a 2-h time period. Superoxide anions, H(2)O(2) and gallic acid quinones were produced during gallic acid autoxidation. In rings partially precontracted with phenylephrine, 0.1-3 microM gallic acid induced marked and largely endothelium-dependent contractions, 10-30 microM gallic acid induced endothelium-independent contractions and 0.1-0.3 mM gallic acid induced complete, fast-developing, endothelium-independent relaxations. Superoxide dismutase (SOD) shifted the endothelium-dependent gallic acid contractions to the right, and N(G)-nitro-l-arginine abolished them. Indomethacin suppressed the endothelium-independent gallic acid contractions, and catalase abolished the endothelium-independent contractions and relaxations. Gallic acid (30 microM) inhibited the relaxant effects of acetylcholine and sodium nitroprusside. In rings maximally precontracted with KCl, 0.1-100 microM gallic acid did not modify the tone, whereas 0.3 mM induced complete, slow-developing, endothelium-independent relaxations. Moreover, 0.3 mM gallic acid induced an irreversible impairment of ring reactivity and the release of lactate dehydrogenase. Catalase and N-acetyl cysteine suppressed the deleterious effects induced by gallic acid in the rings. IN CONCLUSION (a) gallic acid is rapidly and nonenzymatically oxidized in physiological solutions, generating superoxide anions, H(2)O(2) and quinones; (b) superoxide anions (by destroying NO) and low H(2)O(2) levels (by activating cyclooxygenase) both increase vascular tone; (c) moderate H(2)O(2) levels decrease vascular tone; (d) high H(2)O(2) and quinone levels cause irreversible relaxations due to cellular damage.

Study of in vivo and in vitro resting vasodilator nitric oxide tone in normotensive and genetically hypertensive rats

The effects of NG-nitro-L-arginine (L-NNA) on mean arterial pressure and the effects of both L-NNA and methylene blue on isolated aorta tone, were studied in order to elucidate potential alterations in vasodilator resting nitric oxide (NO) tone in genetic hypertension. L-NNA produced a significantly greater increase of mean arterial pressure in spontaneously hypertensive rats (SHR) than in Wistar Kyoto (WKY) rats; in both cases, L-arginine completely inhibited the L-NNA hypertensive effect. Neither ganglion blockade with hexamethonium nor cyclooxygenase inhibition with indomethacin significantly modified the effect of L-NNA in both rat strains. In intact aorta rings, after submaximally contraction with KCI (25 mM), both L-NNA and methylene blue induced strong dose-dependent contractions. The maximum contractions were, however, significantly greater in WKY rats than in SHR. The mechanical elimination of endothelium markedly inhibited both L-NNA and methylene blue maximum contractions. In intact rings, L-arginine completely inhibited the L-NNA effects in both rat strains; in rubbed rings, the L-arginine inhibitory effects were strong in WKY rats but not important and erratic in SHR. L-Arginine had no effect on the contractions induced only by KCI in any of the preparations. In WKY rat-rubbed rings, sodium nitroprusside was significantly more effective in relaxing the contractions in response to 25 mM KCI than the contractions in response to methylene blue. These results indicate that contractions induced by L-NNA and methylene blue in isolated aorta are principally due to the inhibition of an important endothelial resting vasodilator NO tone. They also show that hypertension reduces the resting vasodilator NO tone in isolated rat aorta, in spite of enhancing the total vasodilator NO tone in anaesthetized rat.

G proteins in aortic endothelial cells and bradykinin-induced formation of nitric oxide.

In bovine aortic endothelial cells (BAEC), pertussis toxin (PTx) ADP-ribosylated two major substrates with apparent molecular masses of 40 and 41 kDa, whereas cholera toxin (CTx) ADP-ribosylated two other substrates of 44 and 50 kDa. [alpha-32P]GTP bound to three bands in the 22-27 kDa range. Immunoblot analysis revealed the simultaneous presence of G alpha i1, G alpha i2, G alpha i3, G alpha q or G alpha 11 and of different forms of G alpha s but did not detect significant levels of G alpha 0. Bradykinin caused a 9-fold increase in intracellular cyclic GMP level in BAEC (measured as an index of NO production). Preincubation of BAEC with CTx, but not with PTx, inhibited bradykinin-dependent production of cyclic GMP. These results show that G alpha s, G alpha q or alpha 11, Gi and small GTP-binding proteins are present in BAEC and suggest that a CTx-sensitive G-protein (possibly either small G-protein, G alpha q or G alpha 11) could be associated with the bradykinin-mediated NO formation.

Comparison of the effects of hydralazine and nifedipine on contractions and 45Ca influx of rat aorta

Abstract— The effect of the vasodilator hydralazine has been compared with nifedipine on KCl‐(K+) (60 mM) and noradrenaline‐(NA) (10 μM) induced 45Ca uptake and contractile responses in rat aorta arterial strips without endothelium. Hydralazine (0.5–10 mM) was equally effective in relaxing K+ ‐ (IC50 = 2.2 ± 0.17 mM) and NA‐(IC50 = 3.06 ± 0.25 mM) induced tension, the degree of relaxation depending on the dose. Nifedipine totally inhibited K+‐(IC50 = 3.16 ± 0.28 nM) induced contractions with lower doses than were necessary to relax (up to 54.0 ± 4.1% with supramaximal concentrations) NA‐induced contractions (IC50 = 1.48 ± 0.12 μM). In the experiments in a calcium‐free medium, nifedipine (1 μM) had no effect on the NA‐(10 μM) induced contractions whereas hydralazine (1 mM) strongly inhibited them. Nifedipine did not affect the basal uptake of 45Ca but the induced uptakes were reduced to 66.3 ± 3.2% (K+) and 65.5 ± 4.1% (NA) of their basal values. Hydralazine did not affect the basal uptake of 45Ca nor that induced by the two vascoconstrictor agents. These results suggest that nifedipine acts on the cell membrane by blocking the movements of calcium through the voltage‐dependent and receptor‐operated calcium channels, whilst hydralazine has an intracellular effect.

Role of the endothelial system in Bay-K-8644 enantiomer and nifedipine vasomodulator action in rat aorta

The potential importance of the endothelial system in regulating the effects of (-)-Bay K 8644 (0.1 microM), (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) on resting tension, on contractile responses to noradrenaline (NA) and Ca2+ (in a Ca(2+)-free high-K+ solution), and on basal, NA-induced and K(+)-induced 45Ca2+ uptake, was investigated in rat aorta rings. Mechanical removal of endothelium considerably potentiated the contractile response induced by NA in standard medium and by Ca2+ in Ca(2+)-free high-K+ (15 mM) medium, but did not modify the response induced by Ca2+ in Ca(2+)-free high-K+ (55 mM) medium or by NA in Ca(2+)-free medium. Furthermore, the basal 45Ca2+ uptake and that induced by NA (10 microM) or KCl (15 and 55 mM) were similar in endothelium-rubbed and intact rings. (-)-Bay K 8644 (0.1 microM) shifted the NA and Ca2+ concentration-response curves to the left with potentiation of the maximal contraction. However, (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) caused a shift to the right, with depression of the maximal contraction. The NA concentration-response curves, and those of Ca2+ in Ca(2+)-free high-K+ (55 mM) medium, were affected by the drugs to similar extents, and were not modified by the presence or absence of endothelial cells. The drugs tested did not affect resting tension. Basal 45Ca2+ uptake was not modified by either nifedipine or the Bay K 8644 enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological study of several effects of hydralazine in the bisected rat vas deferens

We have studied several effects of hydralazine in the bisected rat vas deferens. Hydralazine produced a shift to the left of the concentration-response curve for noradrenaline, with potentiation of the maximal response in both portions of the vas deferens. In contrast it caused a shift to the right of the concentration-response curve for noradrenaline in preparations pretreated with cocaine (inhibitor of catecholamine neuronal uptake), and of the curve for methoxamine and for CaCl2 (in depolarizing medium with K+ 55 mM), in all cases with depression of the maximal response. Hydralazine enhanced the contractions induced by noradrenaline in Ca(2+)-free medium, except in the presence of cocaine. It had no effect on [3H]noradrenaline neuronal uptake into noradrenergic neurons of the vas deferens, nor did it affect basal or K(+)-induced 45Ca2+ uptake. These results suggest that hydralazine potentiates the contractions elicited by noradrenaline by a mechanism other than blockade of the neuronal uptake of this catecholamine. Our results also suggest that the inhibition by hydralazine of the contractions elicited by Ca2+ (in Ca(2+)-free depolarizing high-K+ 55 mM solution) and by methoxamine is not due to an action on voltage-dependent Ca2+ channels, but may reflect an intracellular site of action.

Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction

AIMS We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). MAIN METHODS Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. KEY FINDINGS In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. SIGNIFICANCE Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation.

Sildenafil (Viagra®) Prevents Cox-1/ TXA2 Pathway-Mediated Vascular Hypercontractility in ApoE-/- Mice

Background/Aims: The atherosclerotic apolipoprotein E-deficient (apoE-/-) mouse exhibits impaired vasodilation and enhanced vasoconstriction responsiveness. The objectives of this study were: a) to determine the relative contribution of cyclooxygenases (Cox-1 and Cox-2), thromboxane A2 (TXA2) and endothelin-1 (ET-1) to enhancing vascular hyperresponsiveness in this model of atherosclerosis and b) to investigate the beneficial effects of the phosphodiesterase 5 inhibitor sildenafil on this endothelial dysfunction. Methods: Adult male apoE-/- mice were treated with sildenafil (40 mg/kg/day, for 3 weeks) and compared with non-treated ApoE-/- and wild-type mice. The beneficial effects of sildenafil on vascular contractile response to phenylephrine (PE) in aortic rings were evaluated before and after incubation with Cox-1 (SC-560) or Cox-2 (NS-398) inhibitors or the TP antagonist SQ-29548, and on contractile responsiveness to ET-1. Results: ApoE-/- mice exhibited enhanced vasoconstriction to PE (Rmax ∼35%, p<0.01), which was prevented by treatment with sildenafil. The enhanced PE-induced contractions were abolished by both Cox-1 inhibition and TP antagonist, but were not modified by Cox-2 inhibition. Aortic rings from ApoE-/- mice also exhibited enhanced contractions to ET-1 (Rmax ∼30%, p<0.01), which were attenuated in sildenafil-treated ApoE-/- mice. In addition, we observed augmented levels of vascular proinflammatory cytokines in ApoE-/- mice, which were partially corrected by treatment with sildenafil (IL-6, IL-10/IL-6 ratio and MCP-1). Conclusion: The present data show that the Cox-1/TXA2 pathway prevails over the Cox-2 isoform in the mediation of vascular hypercontractility observed in apoE-/-mice. The results also show a beneficial effect of sildenafil on this endothelial dysfunction and on the proinflammatory cytokines in atherosclerotic animals, opening new perspectives for the treatment of other endothelium-related cardiovascular abnormalities.