Ouabain augments Ca(2+) transients in arterial smooth muscle without raising cytosolic Na(+)

Area of Science:

• Cardiovascular Physiology
• Cellular Signaling
• Ion Transport

Background:

• Cardiotonic steroids (CTS) are known to inhibit Na(+) pumps, traditionally thought to increase cytosolic Na(+) and Ca(2+) to affect cardiovascular function.
• This traditional view is challenged by evidence suggesting low-dose CTS may act without significantly altering bulk cytosolic Na(+) concentration ([Na(+)](cyt)).
• The presence of multiple Na(+) pump isoforms in cells also necessitates a re-examination of CTS mechanisms.

Purpose of the Study:

• To investigate the effects of Na(+) pump inhibition on [Na(+)](cyt) and Ca(2+) transients in arterial myocytes.
• To explore the role of localized ion concentrations in cellular signaling pathways.
• To re-evaluate the mechanism of action for cardiotonic steroids at low doses.

Main Methods:

• Primary cultured arterial myocytes were used to measure [Na(+)](cyt) using a Na(+)-binding benzofuran isophthalate indicator.
• Ca(2+) transients were measured using fura 2, a Ca(2+)-sensitive fluorescent dye.
• Experiments involved low concentrations of ouabain, human ouabain-like compound, reduced extracellular K(+), and various ion manipulations (Mg(2+), La(3+)).

Main Results:

• Low concentrations of ouabain (3-100 nM) augmented hormone-evoked Ca(2+) release without increasing bulk [Na(+)](cyt).
• This augmentation was dependent on external Na(+) and inhibited by Mg(2+) and La(3+), suggesting a localized influx mechanism.
• Similar augmentation of Ca(2+) transients was observed in pressurized small resistance arteries, supporting a role in vascular function.

Conclusions:

• Na(+) appears to enter a restricted sub-membrane space between the plasmalemma and sarcoplasmic reticulum via a specific, ion-sensitive mechanism.
• Clusters of high-affinity Na(+) pumps (alpha3) and Na/Ca exchangers in microdomains may control local Na(+) and Ca(2+) concentrations.
• Inhibition of these localized pumps by low-dose ouabain can modulate Ca(2+) signaling, potentially affecting vascular tone and blood flow.