Elena Urcelay

Enhanced Proliferation of Lymphoblasts from Patients with Alzheimer Dementia Associated with Calmodulin-Dependent Activation of the Na+/H+ Exchanger

We have recently reported that lymphoblasts from late onset Alzheimers disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.

Ca2+/calmodulin-dependent modulation of cell cycle elements pRb and p27kip1 involved in the enhanced proliferation of lymphoblasts from patients with Alzheimer dementia.

Failure of cell cycle regulation in neurons might be critically involved in the process of neurodegeneration in Alzheimers disease (AD). We present here evidence to support the hypothesis that cell cycle alterations occur in cells other than neurons in AD sufferers. Lymphocytes from AD patients immortalized with Epstein-Barr virus showed an enhanced rate of proliferation and increased phosphorylation of the retinoblastoma protein (pRb) and other members of the family of pocket proteins compared with cell lines derived from normal age-matched controls. The calmodulin antagonist calmidazolium, as well as W-7 and W-13, abrogated the enhanced activity of AD cells without altering the normal basal rate of proliferation. The effect of calmidazolium was accompanied by partially dephosphorylation of pRb. No changes were found in the expression levels of the G1 cyclin/Cdks complexes. However, lymphoblasts derived from AD patients showed reduced levels of the Cdk inhibitor p27(kip1), which were restored after anti-calmodulin treatment of the cultures. These observations suggest that in AD cells the enhanced rates of cell proliferation and phosphorylation of pRb and the intracellular content of p27(kip1) may be interrelated events controlled by a mechanism dependent on the Ca(2+)/calmodulin signaling pathway. The distinct functional features of lymphoblastoid cells from AD patients offer an invaluable, noninvasive tool to investigate the etiopathogenesis, and eventually, for the early diagnosis and prognosis of this devastating disease.

Characterization of the α1-adrenoceptor-mediated responses in perfused rat liver

Abstract The present work aimed to further characterise the hepatic α1-adrenergic actions by studying the influence of nutritional status and/or extracellular medium composition in the α1-adrenoceptor-induced responses. The experiments were performed in a non-recirculating liver-perfusion system featuring continuous monitoring of vascular resistance, as well as the effluent perfusate changes in pO2, pCa2+, pK+ and pH. The α1-adrenoceptor activation produced biphasic responses to most parameters studied. The acute phase lasted for about 3 min and it was followed by a phase of sustained stimulation that lasted as long as the receptor activation was maintained. Our data indicate that there is not a single pattern of α1-adrenergic responses but variable patterns depending on the nutritional status and the experimental conditions. Gluconeogenic substrates alone produced reciprocal changes in the outflow perfusate pH and Ca2+ activity. The magnitude of these changes indicates that the diversity of α1-adrenoceptor responses are the result of the superposed effects of different rates of substrates and/or metabolites transport. The sustained α1-adrenoceptor stimulation produced extracellular acidification and increases in respiration, vascular resistance and Ca2+ release. These responses required physiological extracellular [Ca2+]. At low extracellular [Ca2+], the α1-adrenoceptor activation failed to acidify the extracellular medium, suggesting that receptor-induced H+ efflux demands normal rates of Ca2+ influx. The correlation between α1-adrenergic-induced increase in O2 uptake and Ca2+ release indicates that the increased energy production can be accounted for by the energy cost of Ca2+ release. The α1-agonist concentration-response studies have shown significant differences in the [α1-agonist]0.5 for each type of response, suggesting the existence of multiple α1-adrenoceptor-coupled signal-transduction pathways.

Modulation of the hepatic α1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca2+-dependent and independent responses

1 . The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic α1‐adrenoceptor‐mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the α1‐agonist‐induced stimulation of gluconeogenesis or phosphorylase ‘a’ activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic α2‐adrenoceptor‐mediated effects indicates that its actions are α1‐specific. 2 . Colchicine enhances the acute α1‐adrenoceptor‐mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the α1‐adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3 . The lack of effect of colchicine in altering the α1‐adrenoceptor ligand binding affinity suggests that it might interact with some receptor‐coupled regulatory element(s). 4 . The acuteness of the colchicine effect and the ability of its isomer β‐lumicolchicine to prevent all the α1‐adrenoceptor‐mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5 . Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin‐induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6 . It is concluded that there are at least two major α1‐adrenoceptor signalling pathways. One is colchicine‐sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C‐independent; the other one is colchicine‐insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C‐independent.

Effect of phenylarsine oxide on hepatic α1-adrenoreceptor responsiveness. dissociation between ionotropic and metabolic responses

The present studies analyze the effect of the tervalent arsenical compound phenylarsine oxide (PAO) on hepatic response to alpha 1-adrenoreceptor stimulation. PAO, while not significantly altering the rate of glycogen breakdown, was found to inhibit many characteristic alpha 1-adrenoreceptor mediated responses including H+ and Ca2+ release, increased energy production, and vascular smooth muscle contraction. PAO inhibited basal gluconeogenesis but failed to inhibit the alpha 1-agonist induced stimulation of glucose production. These data suggest that alpha 1-adrenoreceptor mediated stimulation of metabolism and rates of ion flux across the plasma membrane are separate processes and that exchange in ion homeostasis is not essential to elicit the receptor-mediated metabolic responses. The selective effect of PAO offers an interesting tool for studying the alpha 1-adrenoreceptor signaling mechanisms.