Biomedical subjects

D Guerini

Publications and source records attributed to D Guerini.

At least 19 recordsLinked to original sources

PubMed2002

Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis.

Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are central mediators of the apoptotic program. Here we demonstrate that caspases cleave and inactivate the plasma membrane Ca(2+) pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca(2+) handling, which results in Ca(2+) overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca(2+) handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.

Animals↗

PubMed2000

Single amino acid mutations in transmembrane domain 5 confer to the plasma membrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum.

Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca(2+) pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca(2+) by the sarcoplasmic reticulum Ca(2+) pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La(3+) sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca(2+) binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump.

Animals↗

PubMed2000

Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons.

The Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase export Ca(2+) from the cytosol to the extracellular space. Three NCX genes (NCX1, NCX2, and NCX3), encoding proteins with very similar properties, are expressed at different levels in tissues. Essentially, no information is available on the mechanisms that regulate their expression. Specific antibodies have been prepared and used to explore the expression of NCX1 and NCX2 in rat cerebellum. The expression of NCX2 became strongly up-regulated during development, whereas comparatively minor effects were seen for NCX1. This was also observed in cultured granule cells induced to mature in physiological concentrations of potassium. By contrast, higher K(+) concentrations, which induce partial depolarization of the plasma membrane and promote the influx of Ca(2+), caused the complete disappearance of NCX2. Reverse transcription-polymerase chain reaction analysis showed that the process occurred at the transcriptional level and depended on the activation of the Ca(2+) calmodulin-dependent protein phosphatase, calcineurin. The NCX1 and NCX3 genes were also affected by the depolarizing treatment: the transcription of the latter became up-regulated, and the pattern of expression of the splice variants of the former changed. The effects on the NCX1 and NCX3 genes were calcineurin-independent.

Animals↗

PubMed2000

Calcineurin controls the expression of isoform 4CII of the plasma membrane Ca(2+) pump in neurons.

The expression of the CII splice variant of the plasma membrane Ca(2+) ATPase 4 (PMCA4) was down-regulated in granule neurons when they were cultured under conditions of partial membrane depolarization (25 mM KCl), which are required for long term in vitro survival of the neurons. These conditions, which cause a chronic increase of the resting free Ca(2+) concentration in the neurons, have recently been shown to promote up-regulation of the PMCA2, 3, and 1CII isoforms. Whereas the chronic, i.e. >3 days, Ca(2+) increase was necessary for the up-regulation of the PMCA1CII, 2, and 3, the down-regulation of the PMCA4CII mRNA was already evident 1-2 h after the start of culturing in 25 mM KCl. The immunosuppressant calcineurin inhibitor FK506 inhibited the down-regulation of the PMCA4CII at both the protein and the mRNA level but did not affect the changes of the other PMCA pumps. Direct evidence for the involvement of calcineurin in the down-regulation of the PMCA4CII was obtained by overexpressing a truncated, constitutively active, and Ca(2+)-independent form of calcineurin; under these conditions, depolarization was not required for the down-regulation of the PMCA4CII pump. De novo synthesis of (transcription) factors was required for the down-regulation of the PMCA4CII mRNA. Calcineurin, therefore, controls the neuronal transcription of PMCA4CII, a splice variant of the pump isoforms that is found almost exclusively in brain.

Animals↗

PubMed2000

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.

Animals↗

PubMed1999

Calcium controls the transcription of its own transporters and channels in developing neurons.

Calcium has now become important as a regulator of gene expression. Cerebellar granule cells developing in culture undergo early apoptosis unless their calcium is permitted to increase, e.g., by depolarizing their plasma membrane. The increase is kept within controlled limits by changing the pattern of transcription of calcium transporters: The IP(3) channel (but not the ryanodine channel) becomes strongly up-regulated after some days in culture in a reaction which is controlled by calcineurin. Two plasma membrane calcium pumps (isoforms PMCA2 and PMCA3) also become strongly up-regulated after some days; one (PMCA1) experiences instead a splicing switch which up-regulates a truncated variant of the isoform. By contrast, one splicing variant of the isoform PMCA4 and one of the Na/Ca exchangers of the plasma membrane (NCX2) become very rapidly down-regulated: Their down-regulation is also controlled by calcineurin. The altered pattern of Ca(2+) transporter expression is likely to reflect development-linked changes in the demands for calcium signaling in different domains of the neuronal cell.

Animals↗

PubMed1999

Calcineurin controls inositol 1,4,5-trisphosphate type 1 receptor expression in neurons.

In the central nervous system, release of Ca2+ from intracellular stores contributes to numerous functions, including neurotransmitter release and long-term potentiation and depression. We have investigated the developmental profile and the regulation of inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) in primary cultures of cerebellar granule cells. The expression of both receptor types increases during development. Whereas the expression of type 1 IP3R appears to be regulated by Ca2+ influx through L type channels or N-methyl-D-aspartate (NMDA) receptors, RyR levels increase independently of Ca2+. The main target of Ca2+-influx-regulating IP3R expression is the Ca2+ calmodulin-dependent protein phosphatase calcineurin, because pharmacological blockade of this protein abolishes IP3R expression. Although calcineurin has been shown to regulate the phosphorylation state of the IP3R, the effect described here is at the transcriptional level because IP3R mRNA changes in parallel with protein levels. Thus, calcineurin plays a dual role in IP3R-mediated Ca2+ signaling: it regulates IP3R function by dephosphorylation in the short-term time scale and IP3R expression over more extended periods.

Animals↗

PubMed1999

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+.

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.

Animals↗

PubMed1999

Plasma membrane calcium ATPase isoforms in astrocytes.

The plasma membrane Ca(2+)-ATPase (PMCA) is an essential component of the machinery responsible for cellular Ca(2+) homeostasis. Together with the Na(+)/Ca(2+) exchanger, the plasma membrane Ca(2+)-ATPase (PMCA) is responsible for the extrusion of Ca(2+) from the cytosol. Although both PMCAs and Na(+)/Ca(2+) exchangers are present in high amounts in the brain, it is thought that only the latter localize to glia. This study investigates whether PMCAs are also present in astrocytes and thus are components of Ca(2+) signalling in this cell type. Membrane proteins and mRNA were isolated from primary cultures of rat cortical astrocytes and C6 glioma cells. PMCA isoforms were investigated with isoform specific antibodies and the splice variant pattern was studied in RT-PCR experiments using specific oligonucleotides. The PMCA1, 2, and 4 isoforms were detected in rat cortical astrocytes, whereas only PMCA1 and 2 were found in C6 cells. While neurons express both the CI and CII splice variants, only the splice variant CI of PMCA1, 2, and 4 was detected in astrocytes. Thus, the PMCA pump is present in mammalian glial cells. These results also show that the amounts of PMCA1 and 4 isoforms in astrocytes are comparable to those found in neurons. In contrast, astrocytes contain smaller amounts of PMCA2. Furthermore, PMCA2 and PMCA4 underwent an evident time dependent up-regulation in astrocytes cultured in vitro.

Animals↗

PubMed1998

Protein kinases A and C phosphorylate purified Ca2+-ATPase from rat cortex, cerebellum and hippocampus.

The plasma membrane Ca2+-ATPase (PMCA), the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several independent mechanisms. In this paper we report that the protein kinases A and C differentially activate the Ca2+-ATPase purified from synaptosomal membranes of rat cortex, cerebellum and hippocampus. The effect of protein kinases was more pronounced for the cortical enzyme, whereas cerebellar and hippocampal Ca2+-ATPases were activated to a lesser degree. The preparation of Ca2+-ATPase contained the phosphoamino acids, i.e., P-Ser and P-Thr, indicating that the enzyme was purified in phosphorylated state. The phosphorylation of Ca2+-ATPase by PKA and PKC increased the amount of phosphoamino acids, but in a region-dependent manner. Using the specific antibodies against N-terminal portion of four main PMCA isoforms we have characterized the isoforms composition of Ca2+-ATPase purified from the nervous endings of examined brain areas. Our results indicate that the activity of calcium pump is related to its phosphorylated state, and that the phosphorylation is region-dependent. Moreover, the differences observed could be related to the composition of PMCA isoforms in the different brain areas. Phosphorylation of the plasma membrane Ca2+-ATPase appears to be a mechanism to control its activity. The results support also the possible involvement of PKA and PKC.

Animals↗

PubMed1998

The effect of ethanol on the plasma membrane calcium pump is isoform-specific.

The effect of ethanol has been studied on four different isoforms of the plasma membrane Ca2+-ATPase expressed in Sf9 cells with the help of the baculovirus system. The PMCA2CI protein was maximally activated by 0.5% ethanol, a concentration 8-10 times lower than that needed to obtain the same effect on the PMCA4 protein or on the pump of erythrocyte membranes, which is a mixture of isoforms 1 and 4. Experiments performed with truncated pumps indicated that the stimulation by ethanol was lost if the C-terminal region between Lys1065 and Lys1161, encompassing the calmodulin binding domain, was removed. These observations indicate that the stimulation is the result of a direct interaction of ethanol with the C-terminal regulatory domain of the Ca2+ pump.

Animals↗

PubMed1998

The significance of the isoforms of plasma membrane calcium ATPase.

The plasma membrane calcium ATPase (PMCA) or Ca2+ pump transports Ca2+ ions out of the cells, by using the energy stored in ATP. It is essential in the control of Ca2+ concentration in the cytosol. The plasma membrane Ca2+ pump has been found in all mammalian cells and is encoded by four independent genes. The number of possible isoforms is further increased by alternative splicing at two independent sites; transcripts for more than 20 isoforms have been detected. The PMCA isoforms, in particular some of their alternatively spliced isoforms, have been shown to bind calmodulin with different affinity. The activity of these alternatively spliced pumps is possibly differently regulated by kinase-mediated phosphorylation. A short summary of recent work on the properties of the PMCA isoforms is presented here.

Alternative Splicing↗

PubMed1998

The Ca2+ pumps and the Na+/Ca2+ exchangers.

The Ca2+ ATPases or Ca2+ pumps transport Ca2+ ions out of the cytosol, by using the energy stored in ATP. The Na+/Ca2+ exchanger uses the chemical energy of the Na+ gradient (the Na+ concentration is much higher outside than inside the cell) to remove Ca2+ from the cytosol, Ca2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na+/Ca2+ exchangers are located on the plasma membrane. A Na+/Ca2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca2+ concentration in the cytosol and are vital to prevent Ca2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.

Animals↗

PubMed1998

The calcium pump of the plasma membrane: membrane targeting, calcium binding sites, tissue-specific isoform expression.

The two Ca2+ pumps of higher eucaryotes are strictly targeted to different membrane systems: the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). Chimeric constructs of the two pumps expressed in COS-7 cells have revealed a strong signal for endoplasmic reticulum retention in the N-terminal cytosolic portion of the SERCA pump: the signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention signal is contained in a cytosolic C-terminal sequence immediately preceding the calmodulin-binding domain of the Ca2+ pump. Selective mutations on the SERCA pump have led to the conclusion that 5 conserved residue membrane domains (TM)4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal any of these residues in TM5 of the PMCA pump. Mutation of the conserved residue in TM4 and of two in TM6 abolished the ability of the pump to form the Ca(2+)-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the pump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primary cultures of cerebellar granule cells from newborn rats did not express isoforms 2 and 3 at plating time. Incubation of the cells in depolarizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced variant of isoform 1. Incubation of the cells in L-type Ca2+ channel blockers abolished the upregulation of the pump genes.

Amino Acid Sequence↗

PubMed1997

Calcineurin: not just a simple protein phosphatase.

Calcineurin is a Ca2+ calmodulin dependent protein phosphatase which has an important role in the control of intracellular Ca2+ signalling. The protein is a heterodimer of one catalytic (CnA) subunit and one regulatory (CnB) subunit. As suggested by the protein sequence and confirmed by the crystallographic structure, the catalytic subunit of calcineurin (CnA) has high homologies with other protein phosphatases. The regulatory subunit (CnB) belongs to the EF-hand Ca2+ binding protein family. Despite its similarity with calmodulin, it has a different tertiary structure. Calcineurin is the target of two important immunosuppressant drugs: cyclosporin A and FK506. Subsequently, a detailed clarification of the role of calcineurin in the cytokine mediated activation of the T-cells has been possible. The understanding of the role of calcineurin in other cells, in particular neurons, is also progressing rapidly.

Animals↗

PubMed1997

Immunolocalization of the plasma membrane Ca2+ pump isoforms in the rat brain.

Ca2+ homeostasis in nerve cells is dependent on at least three mechanisms: Ca2+ channels, calcium-binding proteins and Ca2+ exchangers/pumps. Only limited information is available on the regional/cellular distribution of these Ca2+-regulating systems in the brain. The distribution of three of the isoforms of one of the systems, plasma membrane Ca2+-ATPase (PMCA), was analyzed in this study. Using antibodies against epitopes specific for each isoform, a map of the distribution of the pump in the whole brain was produced. The pump was mainly expressed in neurons and was apparently absent from glia cells. Isoform 1 was ubiquitous and occurred in varying, but always significant, concentrations in almost all nerve cells. Isoform 2 was abundant in cerebellar Purkinje cells but less concentrated in other brain regions. Isoform 3 had a predominantly extra neuronal location, e.g. it was abundant in the choroid plexuses. The three isoforms were found to be distributed in a highly characteristic manner, suggesting that nerve cells have different requirements for the preservation of their intracellular calcium homeostasis.

Animals↗

PubMed1996

The plasma membrane calcium pump: recent developments and future perspectives.

The Ca2+ pump of the plasma membrane (PMCA) is regulated by a number of agents. The most important is calmodulin (CaM), which binds to a domain located in the C-terminal portion of the pump, removing it from an autoinhibitory site next to the active site. The CaM-binding domain is preceded by an acidic sequence which contains a hidden signal for endoplasmic reticulum (ER) retention. Chimeras of the PMCA and endoplasmic reticulum (SERCA) pumps have revealed the presence of a strong signal for ER retention in the first 45 residues of the SERCA pump. Four gene products of the PMCA pump are known: two of them (1 and 4) are ubiquitously expressed, two (2 and 3) are specific for nerve cells and may be induced by their activation. Mutagenesis work has identified four residues in three of the transmembrane domains of the pump which may be components of the trans-protein Ca2+ path. The mutation of two of these residues alters the membrane targeting of the pump.

Amino Acid Sequence↗

PubMed1996

Primary structure of human plasma membrane Ca(2+)-ATPase isoform 3.

The complete coding sequence of the human plasma membrane calcium ATPase (PMCA) isoform 3 was determined from overlapping genomic and cDNA clones. The cDNAs for the two major alternative splice variants 3a (3CII) and 3b (3CI) code for proteins of 1173 and 1220 amino-acid residues, respectively, which show 98% identity with the corresponding rat isoforms. On a multiple human tissue Northern blot, a major PMCA3 transcript of about 7 kb was detected exclusively in the brain, demonstrating the highly restricted pattern of expression of this isoform to human neuronal tissues. With the elucidation of the human PMCA3 primary structure, complete sequence information is now available for the entire family of human PMCA isoforms.

Alternative Splicing↗