Magdalena P. Cortés

Calcium signals in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.

Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones.

Alterations of the cationic permeability of red blood cell membranes induced by the photosensitiser nalidixic acid were demonstrated by evaluating the potassium loss from intact erythrocytes. The results show that an increase in intracellular potassium efflux, precedes the photohemolysis induced by nalidixic acid. The addition of a nonpermeable osmotic solute, such as sucrose, inhibited photohemolysis but not the potassium loss, indicating a colloid osmotic lysis. Lipid peroxidation induced by nalidixic acid and other photosensitiser quinolones (oxolinic acid and rosoxacin) was time irradiation-dependent. Although rosoxacin was the most photoperoxidative, none of the three quinolones studied produced significant lipid peroxidation. However, of the three quinolones studied, only rosoxacin considerably diminished the percentage of the cholesterol extracted from red blood cell membranes. It is postulated that the increased cation permeability induced by nalidixic and oxolinic acids cannot be attributed to cholesterol oxidation nor to lipid peroxidation; a more probable mechanism is photo-oxidation of amino acid residues of the membrane proteins. However, the lysis induced by rosoxacin is caused by photo-oxidation of cholesterol, not excluding other cellular targets.

Catecholamines-evoked cytosolic ca2+rise in endothelial cells from bovine adrenal medulla

The effects of catecholamines on intracellular Ca2+concentrations ([Ca2+]i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 μm epinephrine or norepinephrine induced a biphasic [Ca2+]i rise with an initial peak followed by a delayed phase. 10 μm phenylephrine (α1-adrenergic agonist) caused a [Ca2+]i rise similar to that evoked by catecholamines. The increase in [Ca2+]i induced by 10 μm phenylephrine was reverted by 10 μm phenoxybenzamine (α-adrenergic antagonist). Neither isoproterenol (β-adrenergic agonist) nor clonidine (α2-adrenergic agonist) induced [Ca2+]i rise. The initial peak was insensitive to zero external Ca2+ and it was abolished after Ca2+ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca2+ removal. These results indicate that BAMECs possess α1-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca2+ stores and the entry of extracellular Ca2+ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca2+]i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999)

A New Isoxazolic Compound Acts as α7 Nicotinic Receptor Agonist in Human Umbilical Vein Endothelial Cells

Recent evidence suggests that the α7 nicotinic acetylcholine receptors (α7 nAChRs) participate in the development of angiogenesis and could be a new endothelial target for revascularization in therapeutic angiogenesis. It has been shown that in human umbilical vein endothelial cells (HUVECs) α7 nAChR agonists increase the intracellular calcium concentration ([Ca2+]i), thus inducing proliferation and vessel formation which are important stages of angiogenesis. In the present study we evaluated the effect of new isoxazole compounds on the cytosolic Ca2+ signal in HUVECs using the fluorescent Ca2+ indicator Fluo-3AM and probing the involvement of α7 nAChR by means of pharmacological tools. HUVECs expressed mainly α7 nAChR, since there was no significant difference in the increase in [Ca2+]i induced by nicotine, a non-selective nicotinic agonist, in relation to choline, a selective α7 nAChR agonist. The increase in [Ca2+]i induced by 1 mM choline was inhibited significantly (p = 0.014) in cells which had been pre-incubated for 15 min with methyllycaconitine (MLA), a selective α7 nAChR antagonist. The studied compounds 1, 2, and 3 induced an increase in [Ca2+]i in a dose-dependent manner. Compound 1 at 10 mM induced a greater increase in [Ca2+]i than compounds 2 and 3. The increase in [Ca2+]i induced by compound 1 was significantly inhibited by MLA (p = 0.013) and completely inhibited by mecamylamine, a non-selective nAChR antagonist, indicating that the isoxazolic compound 1 acts as an α7 nAChR agonist.

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca(2+) dependent exocitosis of vasoactive compounds. Using the Ca(2+) indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca(2+) increase and depolarization of the membrane potential (20.3+/-0.9 mV; n=23). The increase in cytosolic Ca(2+) induced by 10microM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 microM of hexamethonium. On the other hand, the increase in cytosolic Ca(2+) could be depressed by nifedipine (0.01 -100 microM) or withdrawal of extracellular Ca(2+). Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal glands microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca(2+) channels, influx of external Ca(2+) and liberation of vasoactive compounds.

Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla

We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca2+]i) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca2+]i changes in BAMCECs the intracellular fluorescent probe, fluo‐3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca2+]i, which was depressed by B1650 (0.1 µM), a B2‐bradykinin receptor antagonist (D‐Arg‐[Hyp3, Thi5,8, D‐Phe7]‐Bradykinin). Similarly, increase in [Ca2+]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1‐histamine receptor antagonist. [Ca2+]i increase induced by both agonists was unaffected in the absence of extracellular Ca2+ or presence of antagonists of voltage operated Ca2+ channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca2+]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca2+]i response induced by bradykinin (3 µM), but did not affect the [Ca2+]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin‐ and H1 histamine‐receptors in BAMCECs. Liberation of Ca2+ induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca2+‐ATPase (SERCA) thapsigargin‐insensitive, histamine activates a SER containing a SERCA thapsigargin‐sensitive. We suggest that the increase in [Ca2+]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

Phototoxicity induced by nalidixic and oxolinic acids: Decrease in cell survival of chick embryo fibroblasts and Hep-2 cells

The phototoxic effects of nalidixic and oxolinic acids were evaluated in two types of cultured cells: chick embryo fibroblast and Hep-2 (human laryngo carcinoma cell line). In order to evaluate the phototoxicity induced by nalidixic and oxolinic acids, both cell types were irradiated for 5 min in the presence of each drug. The results showed an inverse relationship between cell survival and the concentration of the drug added to the culture medium. The concentrations of nalidixic and oxolinic acids necessary to induce a phototoxic effect were in the range of therapeutic blood levels. Both chick embryo fibroblasts and Hep-2 cells were more sensitive to the phototoxic effect induced by nalidixic acid than oxolinic acid.

Monoclonal Gammopathy of Undetermined Significance

Monoclonal gammopathies are a heterogeneous group of disorders characterized by the stable or progressive proliferation of an abnormal clone of plasma cells that continue pro‐ ducing antibodies [4]. But because these immunoglobulin proteins are abnormal and mon‐ oclonal (identical copies of each other), these offer no protection against infections and can damage the kidney. This monoclonal immunoglobulin is called M-protein. Each basic unit is a monomeric immunoglobulin consisting of two heavy chains of the same class and subclass and two light chains of the same type. The heavy chain classes are G, A, M, D, E (gamma, alpha, mu, delta, epsilon), while the light chain types are kappa (κ) and lambda (λ).