Effects of Antisense Oligonucleotides to the Cardiac Na+/Ca2+ Exchanger on Calcium Dynamics in Cultured Cardiac Myocytes

Intraneuronal calcium ([Ca 2+ ] i ) regulation is altered in aging brain, possibly because of the changes in critical Ca 2+ transporters. We previously reported that the levels of the plasma membrane Ca 2+-ATPase (PMCA) and the V max for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA i techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro -transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h.

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“…Changes in free [Ca 2+ ] i were determined using fura 2 essentially as we have previously described (Takahashi et al. 1999).…”

Section: Methodsmentioning

confidence: 99%

“…Changes in free [Ca 2+ ] i were determined using fura 2 essentially as we have previously described (Takahashi et al 1999). Hippocampal neurons were used in the Ca 2+ imaging studies because of their greater homogeneity and larger size.…”

Section: Measurement Of Pmca Activity and Intracellular Ca 2+ Regulationmentioning

confidence: 99%

RNA_i– induced silencing of the plasma membrane Ca²⁺– ATPase 2 in neuronal cells: effects on Ca²⁺ homeostasis and cell viability

Fernandes¹,

Zaidi

Bean

et al. 2007

Journal of Neurochemistry

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“…On the other hand, Takahashi et al 1999, using a concentration of 10 μM reported a decrease in the level of NCX1 expression in the order of 20% within 24h [38].…”

Section: Comparison With Previous Studiesmentioning

confidence: 97%

Rapid turnover of the “functional” Na+–Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach

Egger¹,

Porzig²,

Niggli³

et al. 2005

Cell Calcium

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Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na(+)-Ca2+ exchanger (NCX1) to correlate suppression of the Na(+)-Ca2+ exchange function with down-regulation of NCX1 protein expression. In a de-novo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.

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The Cardiac Na⁺‐Ca²⁺Exchanger

Hryshko

2002

Comprehensive Physiology

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Effects of Antisense Oligonucleotides to the Cardiac Na+/Ca2+ Exchanger on Calcium Dynamics in Cultured Cardiac Myocytes

Cited by 8 publications

References 28 publications

RNA_i– induced silencing of the plasma membrane Ca²⁺– ATPase 2 in neuronal cells: effects on Ca²⁺ homeostasis and cell viability

RNA_i– induced silencing of the plasma membrane Ca²⁺– ATPase 2 in neuronal cells: effects on Ca²⁺ homeostasis and cell viability

Rapid turnover of the “functional” Na+–Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach

The Cardiac Na⁺‐Ca²⁺Exchanger

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Effects of Antisense Oligonucleotides to the Cardiac Na+/Ca2+ Exchanger on Calcium Dynamics in Cultured Cardiac Myocytes

Cited by 8 publications

References 28 publications

RNAi– induced silencing of the plasma membrane Ca2+– ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

RNAi– induced silencing of the plasma membrane Ca2+– ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Rapid turnover of the “functional” Na+–Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach

The Cardiac Na+‐Ca2+Exchanger

Contact Info

Product

Resources

About

RNA_i– induced silencing of the plasma membrane Ca²⁺– ATPase 2 in neuronal cells: effects on Ca²⁺ homeostasis and cell viability

RNA_i– induced silencing of the plasma membrane Ca²⁺– ATPase 2 in neuronal cells: effects on Ca²⁺ homeostasis and cell viability

The Cardiac Na⁺‐Ca²⁺Exchanger