Manuel Campos-Toimil

Study of the mechanisms involved in the vasorelaxation induced by (−)-epigallocatechin-3-gallate in rat aorta

This study investigated several mechanisms involved in the vasorelaxant effects of (−)‐epigallocatechin‐3‐gallate (EGCG). EGCG (1 μM–1 mM) concentration dependently relaxed, after a transient increase in tension, contractions induced by noradrenaline (NA, 1 μM), high extracellular KCl (60 mM), or phorbol 12‐myristate 13‐acetate (PMA, 1 μM) in intact rat aortic rings. In a Ca2+‐free solution, EGCG (1 μM–1 mM) relaxed 1 μM PMA‐induced contractions, without previous transient contraction. However, EGCG (1 μM–1 mM) did not affect the 1 μM okadaic acid‐induced contractions. Removal of endothelium and/or pretreatment with glibenclamide (10 μM), tetraethylammonium (2 mM) or charybdotoxin (100 nM) plus apamin (500 nM) did not modify the vasorelaxant effects of EGCG. In addition, EGCG noncompetitively antagonized the contractions induced by NA (in 1.5 mM Ca2+‐containing solution) and Ca2+ (in depolarizing Ca2+‐free high KCl 60 mM solution). In rat aortic smooth muscle cells (RASMC), EGCG (100 μM) reduced increases in cytosolic free Ca2+ concentration ([Ca2+]i) induced by angiotensin II (ANG II, 100 nM) and KCl (60 mM) in 1.5 mM CaCl2‐containing solution and by ANG II (100 nM) in the absence of extracellular Ca2+. In RASMC, EGCG (100 μM) did not modify basal generation of cAMP or cGMP, but significantly reversed the inhibitory effects of NA (1 μM) and high KCl (60 mM) on cAMP and cGMP production. EGCG inhibited the enzymatic activity of all the cyclic nucleotide PDE isoenzymes present in vascular tissue, being more effective on PDE2 (IC50∼17) and on PDE1 (IC50∼25). Our results suggest that the vasorelaxant effects of EGCG in rat aorta are mediated, at least in part, by an inhibition of PDE activity, and the subsequent increase in cyclic nucleotide levels in RASMC, which, in turn, can reduce agonist‐ or high KCl concentration‐induced increases in [Ca2+]i.

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.

Synthesis and vasorelaxant activity of new coumarin and furocoumarin derivatives.

We have synthesized a new series of coumarins and furocoumarins and evaluated their vasorelaxant activity in rat aorta rings pre-contracted with noradrenaline or by depolarisation with high KCl. The new furocoumarins relax smooth vascular muscle with a profile similar to that of khellin (a furochromone that directly relaxes smooth muscle) and at a greater potency, suggesting that these compounds have a potential interest for the development of new and more efficient vasodilator drugs.

Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca2+ responses in endothelial cells

Background and purpose: We previously reported that agonist‐induced rises in cytoplasmic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short‐term (2 min) pre‐treatment with cAMP‐elevating agents. The aim of this work was to study the effects of longer term (8 h) pre‐treatment with dibutyryl‐cAMP (db‐cAMP) or rolipram, a specific inhibitor of phosphodiesterase‐4 (PDE4), on [Ca2+]i, cAMP levels and PDE activity and expression in HUVEC.

Real-time studies of zymogen granule exocytosis in intact rat pancreatic acinar cells

1 An adequate understanding of secretion requires the measurement of exocytosis on the same time scale as that used for second messenger dynamics. To investigate the kinetics of ACh‐evoked secretion in pancreatic acinar cells, exocytosis of zymogen granules was quantified by continuous, time‐differential analysis of digital images. The validity of this method was confirmed by simultaneous fluorescence imaging of quinacrine‐loaded zymogen granules. 2 Basal rates of exocytosis were low (0.2 events min−1). ACh stimulated a biphasic increase in secretory activity, maximal rates exceeding 20 events min−1 after 10 s of ACh application (10 μm). Over the next 15 s the rate of exocytosis fell to less than 4 events min−1; then began a second phase of secretion that peaked 15 s later at ∼11 events min−1, but subsequently declined in the continued presence of agonist. 3 Measurements of fura‐2 fluorescence demonstrated a biphasic increase in intracellular [Ca2+] ([Ca2+]i). Comparison of the [Ca2+]i records and time‐differential analysis revealed that the fall in exocytotic rate following the initial burst occurred despite the fact that [Ca2+]i remained high. 4 The second phase of secretion depended on both [Ca2+]i and [ACh]. At 10 μm ACh there was a decrease in the steepness of the relationship between [Ca2+]i and exocytosis that led to an enhancement of the slow secretory phase. 5 We propose that acinar cells contain two pools of secretory vesicles: a small pool of granules that is exocytosed rapidly, but is quickly depleted; and a reserve pool of granules that can be recruited by ACh in a process that is modulated by second messengers other than calcium.

Effects of (−)-epigallocatechin-3-gallate in Ca2+-permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.

Study of the in vivo and in vitro cardiovascular effects of (+)-glaucine and N-carbethoxysecoglaucine in rats.

1 The cardiovascular and vasorelaxant effects of (+)‐glaucine and of a semisynthetic derivative (N‐carbethoxysecoglaucine) were studied in rats.

Synthesis and pharmacological evaluationof 2,3-dihydro-3-oxo-4H-thieno[3,4-e][1,2,4]thiadiazine 1,1-dioxidesas voltage-dependent calcium channel blockers

The synthesis of a novel series of 2,3-dihydro-3-oxo-4H-thieno[3, 4-e][1,2,4]thiadiazine 1,1-dioxides and their pharmacological evaluation as drugs with effects on the rat cardiovascular system are described. The compounds under study were synthesized via Curtius rearrangement of appropriate sulfamoylacylazides which, in turn, were prepared from known starting materials. In isolated rat portal vein, these thienothiadiazines, like verapamil and diazoxide, inhibited the spontaneous motility produced by KCl (20 mM). In addition, the new compounds, like verapamil and unlike diazoxide, also exhibited inhibitory effects in the same preparation when the cell membrane was depolarized by an increased extracellular KCl concentration (80 mM) and, consequently, the membrane potential approached a level close to the K(+) equilibrium potential. Further characterization of this inhibitory activity led to the identification of a selective inhibitory effect of the new compounds on KCl (80 mM)-induced 45Ca(2+) uptake in the same vascular tissue. When tested in vivo (anaesthetized normotensive rats), acute administration of verapamil, diazoxide and some of the most in vitro potent compounds in 45Ca(2+) uptake experiments produced a gradual, dose-dependent and sustained decrease in diastolic arterial blood pressure, devoid of cardiac effects. These results suggest that, like verapamil, the cardiovascular effects produced by the new thienothiadiazines seem to be due, at least in part, to a blockade of transmembrane voltage-dependent calcium channels present in vascular smooth muscle cells and not to an activation of ATP-sensitive K(+) channels. Compounds 5b, 5e and 5i have been selected for further studies as antihypertensive agents.

Pyridazine derivatives XIV. Study of the vasorelaxant action of 6-aryl-5-piperidino-3-hydrazinopyridazines in isolated rat thoracic aorta: Comparison with hydralazine

1. For several years we have been working on the synthesis of modified hydrazinopyridazines which have proved to possess remarkable vasorelaxant and antihypertensive activity. We now report the vasodilator effects of three novel 6-aryl-5-piperidino-3-hydrazinopyridazines (1a, 1b and 1c), structurally related to the well-known antihypertensive drug hydralazine. 2. Hydralazine and the new hydrazinopyridazines relaxed, in a concentration-dependent and nonspecific way, the contractions elicited by noradrenaline or a high K+ concentration in rat aortic rings with or without endothelium. According to the IC50 (50% inhibitory concentrations) values obtained, the vasorelaxant potency of the new compounds was greater than that of hydralazine. 3. In a Ca2+-free medium, the contractions provoked by noradrenaline or caffeine were significantly inhibited by the new hydrazinopyridazines and by hydralazine. 4. Hydralazine and the novel molecules did not significantly modify basal, noradrenaline- or K+-induced 45Ca2+ uptake. 5. These results suggest that 1a, 1b and 1c have an endothelium-independent vasorelaxant activity greater than that of hydralazine in isolated rat aortic rings, which seems not to be mediated by a blockade of transmembrane Ca2+ movements through specific channels. This effect could be due, at least in part, to an intracellular mechanism of action.

Two Modes of Secretion in Pancreatic Acinar Cells: Involvement of Phosphatidylinositol 3-Kinase and Regulation by Capacitative Ca2+ Entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.