Cell swelling increases intracellular calcium in Necturus erythrocytes

Area of Science:

• Cellular Physiology
• Ion Transport Mechanisms
• Erythrocyte Function

Background:

• Regulatory volume decrease (RVD) is vital for cell survival under hypotonic stress.
• Calcium ions (Ca2+) are implicated in various cellular signaling pathways, including volume regulation.

Purpose of the Study:

• To investigate the role of cytosolic free Ca2+ in the RVD of Necturus erythrocytes.
• To determine how hypotonic shock influences intracellular Ca2+ levels and subsequent cell volume changes.

Main Methods:

• Epi-fluorescence microscopy using fluo-4-AM to measure cytosolic Ca2+.
• Hypotonic shock applied to Necturus erythrocytes.
• Manipulation of extracellular and intracellular Ca2+ levels.
• Measurement of osmotic fragility and cell volume recovery (Coulter counter).
• Whole-cell patch clamp electrophysiology.

Main Results:

• Hypotonic shock (50% tonicity) significantly increased cytosolic free Ca2+.
• This Ca2+ increase was dependent on extracellular Ca2+ and inhibited by EGTA, hexokinase, suramin, and gadolinium.
• Low extracellular Ca2+ increased osmotic fragility and reduced the rate of volume recovery.
• Ca2+ ionophore A23187 potentiated volume recovery.
• Low Ca2+ or BAPTA-AM buffering reduced volume recovery rate and inhibited swelling-activated whole-cell currents.

Conclusions:

• Hypotonic shock induces an increase in cytosolic free Ca2+ in Necturus erythrocytes.
• This Ca2+ influx is a critical component of the regulatory volume decrease mechanism.
• Ca2+-dependent ion currents play a significant role in facilitating cell volume recovery.