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Antisense inhibition of Na+/Ca2+ exchange in primary cultured arterial myocytes
M K Slodzinski1, M Juhaszova, M P Blaustein
1Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.
Abstract:
The effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the Na+/Ca2+ exchanger (NCX) were tested in primary cultured rat mesenteric artery myocytes. In parallel cultures, myocytes proliferated and were morphologically normal in the presence of scrambled nonsense (NS-) or AS-oligos or no oligos (controls). NCX function was examined with digital imaging, using fura 2 to estimate the cytosolic free Ca2+ concentration ([Ca2+]cyt). Resting [Ca2+]cyt was higher (145 +/- 4 nM; P < 0.05) in AS-oligo-treated cells than in controls (125 +/- 5 nM) or NS-oligo-treated cells (131 +/- 4 nM). Lowering external Na+, to promote Ca2+ entry via NCX, increased [Ca2+]cyt transiently in controls and NS-oligo-treated cells but not in AS-oligo-treated cells. Raising the cytosolic free Na+ concentration with ouabain augmented the low-Na(+)-induced rise in [Ca2+]cyt in controls and NS-oligo-treated cells, but AS-oligo-treated cells still did not respond. Nevertheless, serotonin (5-HT) increased [Ca2+]cyt in all three groups. Thus AS-oligos selectively blocked NCX activity but not the 5-HT response. To determine the effect of NCX knockdown on the modulation of stored Ca2+, the 5-HT response was tested immediately after removal of external Ca2+. Ouabain augmented the 5-HT-induced rise in [Ca2+]cyt in control and NS-oligo-treated cells but not AS-oligo-treated cells. This indicates that the NCX can modulate intracellular Ca2+ stores. We conclude that AS-oligos are useful for investigating the physiological role of NCX in vascular smooth muscle.
Insights
Antisense oligodeoxynucleotides (AS-oligos) effectively blocked the Na+/Ca2+ exchanger (NCX) in rat artery cells. This selective blockade allows researchers to investigate the NCX
Area of Science:
- Cardiovascular Biology
- Molecular Pharmacology
- Cell Physiology
Background:
- The Na+/Ca2+ exchanger (NCX) plays a critical role in regulating intracellular calcium levels in vascular smooth muscle cells.
- Understanding NCX function is essential for elucidating mechanisms of vascular tone and disease.
- Antisense oligodeoxynucleotides (AS-oligos) offer a targeted approach to modulate gene and protein expression.
Purpose of the Study:
- To investigate the physiological role of the NCX in primary cultured rat mesenteric artery myocytes.
- To determine the efficacy of chimeric phosphorothioated AS-oligos in selectively inhibiting NCX activity.
- To explore the impact of NCX inhibition on intracellular calcium handling and responses to stimuli.
Main Methods:
- Primary cultured rat mesenteric artery myocytes were treated with AS-oligos targeting the NCX, scrambled nonsense oligos, or no oligos (controls).
- Cellular proliferation and morphology were assessed to ensure viability.
- Intracellular calcium concentrations ([Ca2+]cyt) were measured using digital imaging with fura 2.
- NCX function was probed by altering external sodium concentrations and using ouabain to manipulate intracellular sodium.
- Responses to serotonin (5-HT) stimulation were evaluated under various conditions, including calcium-depleted media.
Main Results:
- AS-oligo treatment led to a significant increase in resting [Ca2+]cyt compared to controls.
- AS-oligos selectively blocked NCX-mediated calcium influx induced by low external sodium.
- The 5-HT-induced increase in [Ca2+]cyt was not affected by AS-oligos, indicating specificity.
- NCX inhibition by AS-oligos altered the modulation of intracellular calcium stores, as evidenced by blunted 5-HT responses after calcium removal.
Conclusions:
- Chimeric phosphorothioated AS-oligos are effective tools for selectively inhibiting NCX activity in vascular smooth muscle cells.
- The NCX plays a significant role in modulating intracellular calcium stores.
- AS-oligos provide a valuable method for dissecting the physiological functions of the NCX in cardiovascular research.