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

Expression, purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity.

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms

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

Protein kinases A and C phosphorylate purified Ca2+-ATPase from rat cortex, cerebellum and hippocampus1A preliminary report of the PKA- and PKC-mediated phosphorylation of Ca2+-ATPase purified from rat brain was presented at the FEBS Special Meeting: Cell Signalling Mechanisms, Amsterdam, The Netherlands, June 29–July 3, 1997.1

AbstractThe 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

Calcineurin Controls the Expression of Isoform 4CII of the Plasma Membrane Ca2+ Pump in Neurons *

Abstract The expression of the CII splice variant of the plasma membrane Ca2+ 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 Ca2+ 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, Ca2+ 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 Ca2+-independent form of calcineurin; under these conditions, depolarization was not required for the down-regulation of the PMCA4CII pump. De novosynthesis 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

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo

Excesso de peso e ingestão de baixa qualidade da dieta em pacientes com hipertensão pulmonar : um perfil diferente de paciente com doença pulmonar

Background: Pulmonary hypertension (PH) is characterized by elevated blood pressure in the pulmonary artery. The literature is still scarce about nutritional approaches to this disease. However, is well known that high diet quality has a beneficial impact onquality of life,progression, and mortality of patients with chronic lung diseases, and this may apply to PH as well. Aims: To evaluate diet quality in patients with PH and characterize their comorbidities. Materials and Methods: Cross-sectional study with 35 patients. Body mass index, body fat, food intake, blood biochemical parameters were assessed. Diet quality was evaluated with the Healthy Eating Index (HEI) instrument. Results: The sample consisted predominantly of women (77.2%); 57.1% of the subjects were overweight or obese. Systemic arterial hypertension was the most prevalent comorbidity (28.6%), and one-third of the sample had glycemic changes and hypertriglyceridemia. Most subjects (82.9%) had low diet quality, and none had diet quality classified as good. Intake of fiber, calcium, and monounsaturated fatty acids was below current recommendations, while intake of protein and saturated fatty acids exceeded recommendations (p<0.05). Discussion and Conclusion: This sample of patients with PH was predominantly overweight/obese and had poor diet quality. The presence of chronic non-communicable diseases, altered glucose levels, and hyperlipidemia is consistent with these findings, possibly because of poor diet qualityIntrodução: A Hipertensão Pulmonar (HP) é caracterizada pela elevação da pressão sanguínea na artéria pulmonar. Em relação à nutrição nesta doença a literatura ainda é escassa, porém sabe-se que a dieta de alta qualidade em nutrientes desempenha papel importante na qualidade de vida, progressão e mortalidade de pneumopatias,podendo se aplicar para HP da mesma forma. Objetivos: Avaliar o Índice de alimentação saudável (IAS) de pacientes com HP e conhecer suas comorbidades. Materiais e Métodos: Estudo transversal, realizado com 35 pacientes portadores de HP. Foram avaliados índice de massa corporal, percentual de gordura corporal, exames bioquímicos, além do índice da qualidade da dieta por meio do instrumento IAS. Resultados: A amostra foi composta por 77% indivíduos do sexo feminino e 57,1% eram sobrepesos ou obesos.Hipertensão arterial sistêmica foi a principal comorbidade apresentada (28,6%), além de que 1/3 dos indivíduos possuírem alterações glicêmicas e hipertrigliceridemia. De todos os avaliados, 82,9% apresentaram alimentação de baixa qualidade. A ingestão de fibras, cálcio e ácidos graxos monoinsaturados (AGM) estavam aquém das recomendações vigentes (p<0,05), enquanto a de proteínase ácidos graxos saturados excederam estas mesmas recomendações (p<0,05). Conclusão: A amostra avaliada forampredominantemente de obesos e sobrepesos e tem baixa qualidade da dieta. A presença de doenças crônicas não-transmissíveis, alterações glicêmicas e de triglicerídeos complementam esses achados, possivelmente como consequências dele

A Potent and Selective S1P1 Antagonist with Efficacy in Experimental Autoimmune Encephalomyelitis

SummaryLymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P1), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P1 receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P1 antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P1 antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P1 receptor antagonism and to differentiate the effects from those of S1P1 agonists

A Monoselective Sphingosine-1-Phosphate Receptor-1 Agonist Prevents Allograft Rejection in a Stringent Rat Heart Transplantation Model

SummaryFTY720 is an immunomodulator with demonstrated efficacy in a phase II trial of relapsing multiple sclerosis. FTY720-phosphate, the active metabolite generated upon phosphorylation in vivo, acts as a potent agonist on four of the five known sphingosine-1-phosphate (S1P1) receptors. AUY954, an aminocarboxylate analog of FTY720, is a low nanomolar, monoselective agonist of the S1P1 receptor. Due to its selectivity and pharmacokinetic profile, AUY954 is an excellent pharmacological probe of S1P1-dependent phenomena. Oral administration of AUY954 induces a profound and reversible reduction of circulating lymphocytes and, in combination with RAD001 (Certican/Everolimus, an mTOR inhibitor), is capable of prolonging the survival of cardiac allografts in a stringent rat transplantation model. This demonstrates that a selective agonist of the S1P1 receptor is sufficient to achieve efficacy in an animal model of transplantation