When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time.
Key points• Inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) are functionally expressed in cardiac myocytes.• The influence of inositol 1,4,5-trisphosphate-induced sarcoplasmic reticulum (SR)-Ca 2+ release (IP3ICR) on atrial excitation-contraction coupling (ECC) under physiological and pathophysiological conditions remains elusive.• The present study focuses on local IP3ICR and its functional consequences for ryanodine receptor (RyR) activity and subsequent Ca 2+ -induced Ca 2+ release in atrial myocytes.• Here we show significant SR-Ca 2+ flux, but eventless SR-Ca 2+ release through InsP 3 Rs.• We suggest a new mechanism based on eventless and highly efficient InsP 3 -dependent SR-Ca 2+ flux as a crucial mechanism of functional cross-talk between InsP 3 Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.Abstract Augmented inositol 1,4,5-trisphosphate receptor (InsP 3 R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca 2+ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca 2+ -induced Ca 2+ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP 3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP 3 was accompanied by a significant increase in the Ca 2+ release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm) −1 s −1 ). These individual photolytically triggered Ca 2+ release events were identified as Ca 2+ sparks, which originated from RyR openings. This was verified by Ca 2+ spark analysis and pharmacological separation between RyR and InsP 3 R-dependent sarcoplasmic reticulum (SR)-Ca 2+ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca 2+ flux but eventless SR-Ca 2+ release through InsP 3 R were characterized using SR-Ca 2+ leak/SR-Ca 2+ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca 2+ spark probability. We conclude that eventless and highly efficient InsP 3 -dependent SR-Ca 2+ flux is the main mechanism of functional cross-talk between InsP 3 Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.
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