Angel Nadal

Activation of L‐arginine transport (system y+) and nitric oxide synthase by elevated glucose and insulin in human endothelial cells.

1. Modulation of L‐arginine transport (system y+) and release of nitric oxide (NO) and prostacyclin (PGI2) by elevated glucose and insulin were investigated in human cultured umbilical vein endothelial cells. 2. Elevated glucose induced a time‐ (6‐12 h) and concentration‐dependent stimulation of L‐arginine transport, which was reversible and associated with a 3‐fold increase in intracellular cGMP accumulation (index of NO synthesis) and 75% decrease in PGI2 production. 3. Elevated glucose had no effect on the initial transport rates for L‐serine, L‐citrulline, L‐leucine, L‐cystine or 2‐deoxyglucose. 4. Resting membrane potential was unaffected by elevated glucose whereas basal intracellular [Ca2+] increased from 65 +/‐ 5 nM to 136 +/‐ 16 nM. 5. Insulin induced a protein synthesis‐dependent stimulation of L‐arginine transport and increased NO and PGI2 production in cells exposed to 5 mM glucose. 6. In cells exposed to high glucose, insulin downregulated elevated rates of L‐arginine transport and cGMP accumulation but had no effect on the depressed PGI2 production. 7. Our findings suggest that insulins normal stimulatory action on human endothelial cell vasodilator pathways may be impaired under conditions of sustained hyperglycaemia.

ACTIONS OF SERUM AND PLASMA ALBUMIN ON INTRACELLULAR CA2+ IN HUMAN ENDOTHELIAL CELLS

1 The effects of serum and plasma albumin on [Ca2+]i in human endothelial cells were examined using single‐cell Ca2+ imaging. Two types of endothelial cell were used: human umbilical vein endothelial cells (HUVEC) in primary culture, and the endothelial‐derived cell line ECV304. 2 Serum albumin caused a large and transient rise in [Ca2+]i, due to Ca2+ release from an IP3‐sensitive internal store, followed by a maintained elevation in [Ca2+]i attributable to Ca2+ influx from the external medium. A half‐maximal rise in [Ca2+]i was produced by a concentration of serum albumin of about 1 μg ml−1. 3 The Ca2+‐releasing action of serum albumin is abolished by methanol extraction and is therefore attributable to an attached polar lipid. A possible candidate is lysophosphatidic acid, known to be released from platelets during blood coagulation, which produced similar effects to those of serum albumin. 4 In HUVEC, plasma albumin caused a sustained decrease in [Ca2+]i from the mean resting level of 114 nm to 58 nm. No effect of plasma albumin was observed in ECV304 cells. 5 The decrease in [Ca2+]i caused by plasma albumin is due to an uptake into intracellular stores. The store loading substantially potentiates the action of Ca2+‐releasing agonists such as histamine. 6 The results show that normal plasma albumin, which carries few lipids, lowers [Ca2+]i and potentiates the actions of Ca2+‐releasing agonists by promoting Ca2+ uptake into intracellular stores. When converted to the serum form, by binding lysophosphatidic acid released during blood coagulation, albumin has a potent effect in elevating [Ca2+]i. Blood coagulation may therefore play a role in regulating vascular tone and capillary permeability.

Plasma Albumin Induces Calcium Waves in Rat Cortical Astrocytes

Changes in intracellular calcium were monitored in cultured cortical astrocytes stimulated with albumin. Albumin elicited intracellular calcium mobilisation from intracellular stores, inducing repetitive intracellular calcium oscillations. The oscillations were not blocked by ryanodine, a blocker of the Ca‐induced Ca release mechanism, and the release occurred from the same store as is accessed by glutamate and bradykinin, both of which release calcium by an IP3‐dependent mechanism. Calcium signals induced by albumin appear therefore to occur via a pure IP3‐dependent mechanism. When albumin was applied to confluent monolayers of astrocytes, the oscillations in individual cells were initially unsynchronised, but after several minutes of application, the Ca2+ oscillations were observed to synchronise and spread through the astrocyte network as a wave. These intercellular calcium waves were inhibited by the gap junction blocker halothane. Using the fluorescence recovery after photobleaching (FRAP) technique, we demonstrate that the development of propagated waves with prolonged exposure to albumin does not result from an increase in cell coupling. The development of calcium waves on exposure to albumin may be important in the formation of glial scars in the CNS after breakdown of the blood‐brain barrier. GLIA 19:343–351, 1997.

Albumin stimulates uptake of calcium into subcellular stores in rat cortical astrocytes.

1. When albumin from either plasma or serum is applied at low concentrations to cortical astrocytes a decrease in the level of [Ca2+]i is observed. At higher concentrations trains of calcium spikes are seen. 2. Removal of the polar lipids which are normally bound to native albumin abolishes the ability to induce spikes, but the decrease in [Ca2+]i is unaffected. The decrease is abolished by the denaturation of albumin and is not reproduced by a number of other proteins, and is therefore a specific action of albumin. We conclude that native albumin has a dual agonist action: the decrease in [Ca2+]i is induced by the albumin protein molecule, while the spikes are induced by a lipid normally bound to it. 3. The decrease is rapid (fastest tau = 12 s) and the rate is dependent on the concentration of albumin. [Ca2+]i falls from 77 nM to around 34 nM in the presence of saturating levels of albumin, and this level appears to be maintained indefinitely. 4. The decrease is due to an uptake of calcium into subcellular stores, as it is not abolished by removal of external Ca2+ or Na+ but is abolished by thapsigargin and cyclopiazonic acid, which are specific inhibitors of the endoplasmic reticulum Ca(2+)‐ATPase. 5. When the state of store filling after albumin application is probed with a pulse of glutamate it can be seen that stores fill with the same time course as the decrease in [Ca2+]i. The low level of [Ca2+]i in albumin must therefore be maintained by a suppression of calcium influx rather than by a continued uptake into stores. 6. The calcium uptake potentiates the efficacy of low concentrations of calcium‐releasing agonists such as glutamate and bradykinin by almost an order of magnitude. 7. A possible function for the calcium uptake caused by albumin is to potentiate the production of calcium spike trains by promoting refilling of calcium stores in the intervals between spikes. The uptake may play a role in the response of astrocytes to damage in the CNS.

Albumin elicits calcium signals from astrocytes in brain slices from neonatal rat cortex

1 Albumin causes calcium signals and mitosis in cultured astrocytes, but it has not been established whether astrocytes in intact brain also respond to albumin. The effect of albumin on intracellular calcium concentration ([Ca2+]i) in single cells was therefore studied in acutely isolated cortical brain slices from the neonatal rat. 2 Physiological concentrations of albumin from plasma and from serum produced an increase in [Ca2+]i in a subpopulation of cortical cells. Trains of transient elevations in [Ca2+]i (Ca2+ spikes) were seen in 41 % of these cells. 3 The cells responding to albumin are identified as astrocytes because the neurone‐specific agonist NMDA caused much smaller and slower responses in these cells. On the other hand NMDA‐responsive cells, which are probably neurones, exhibited only small and slow responses to albumin. The residual responses of astrocytes to NMDA and neurones to albumin are likely to be due to crosstalk with adjacent neurones and astrocytes, respectively. 4 Methanol extraction of albumin removes a polar lipid and abolishes the ability of albumin to increase intracellular calcium. 5 Astrocyte calcium signalling caused by albumin may have important physiological consequences when the blood‐brain barrier breaks down and allows albumin to enter the CNS.

Lysophospholipids trigger calcium signals but not DNA synthesis in cortical astrocytes

Astrocytes generate calcium signals and proliferate in response to a growth factor‐like lipid bound to plasma and serum albumin, in a process likely to be important in the formation of glial scars. A number of potential candidates for the physiologically active lipid were investigated. Lysophosphatidic acid, lysophosphatidylcholine, sphingomyelin, and platelet‐activating factor all elicited calcium signals of varying magnitudes in cortical astrocytes, although only lysophosphatidic acid elicited calcium signals comparable in amplitude to those induced by the active physiological lipid. None of these lipids, however, caused cell division in astrocytes. There is therefore no invariable relationship between the ability of lipids to induce calcium signals and mitogenic activity. None of the lipids investigated demonstrate the activity of the natural lipid factor in generating both calcium signals and mitotic activity in astrocytes. GLIA 28:272–276, 1999.

Imaging Intracellular Calcium in Living Tissue by Laser-Scanning Confocal Microscopy

For many years physiologists have attempted to watch cells working within organs and to visualize the interactions amongst the different types of cells within them. The development of fluorescence imaging systems and calcium sensitive fluorescent dyes has essentially contributed to revealing many important aspects of cell signaling. Although it has been proven that conventional fluorescence microscopy is an excellent technique for detecting calcium signals in isolated cells, it only provides a very confined insight when used in tissue. When ordinary fluorescence microscopy is used with relatively thick specimens, the main limitations are due to image degradation caused by out of focus flare, further limited by a poor discrimination of depth. In conventional fluorescence, the light that makes up each point of the image spreads out in a solid cone that can reach a significant distance above and below the focus. The spreading cone of light blurs the focused image of the specimen. Also, fluorescent objects that are out of focus introduce unwanted light which highly reduces the contrast of the image. These problems are greatly reduced using laser-scanning confocal microscopy (LSCM), which eliminates out of focus fluorescence, producing well defined thin optical sections out of thick fluorescence specimens (White et al., 1987); (Fine et al., 1988); (Pawley, 1995). The elimination of out of focus fluorescence increases contrast, clarity and detection, enabling us to do a wide range of investigations that, up until now, have been difficult or impossible to carry out with previous techniques.

Characteristics of a Nonclassical Membrane Estrogen Receptor in the Endocrine Pancreas

Estrogen membrane actions are triggered after estrogens are bound to a variety of membrane proteins. Those include: ion channels, other ligand receptors, and specific plasma membrane estrogen receptors. Some of the estrogen binding sites have different affinities and therefore, respond to distinct estrogen concentrations ([1–[4).