Rundown of reverse mode of Na⁺/Ca²⁺ exchanger (NCX1). (A) When NCX1 was repeatedly stimulated with Na⁺-free, 1 mM Ca²⁺-containing solution, a progressive decrease of the Ca²⁺ transients was observed. A Na⁺-pump inhibitor, ouabain, did not enhance the transients. (B) Increased rising time of the Ca²⁺ transients was observed during rundown. The representative traces recorded from the same cell were drawn for comparison. An arrow indicates the starting time of stimulation. The mean values of the time-to-peak values are described in text. The peak ratio values of the 1^st, 2^nd, and 3^rd Ca²⁺ transient were 3.1±0.44, 2.4±0.25, and 1.8±0.15, respectively (n=4). (C) Rundown of the outward whole-cell NCX1 current. (D) The degrees and the time courses of rundown obtained from the Ca²⁺ transients (n=13) and the whole-cell current (n=7) were compared. Amplitudes of the 2^nd, 3^rd, and 4^th Ca²⁺ transient were reduced to 48±12, 35±9.7, and 28±8.4% of the initial (‘1^st’) transient, respectively. Amplitudes of the 2^nd, 3^rd, and 4^th current were reduced to 20±3.2, 17±2.8, and 16±2.9% of the initial one, respectively.

Using a confocal imaging, changes of the protein kinase C activity and the PIP₂ concentration during the Ca²⁺ transients were monitored via PKCβ -II-GFP and PLCδ1-PH-GFP. Fluorescence intensity obtained along the horizontal axis was shown as histogram with corresponding confocal image. The normalized fluorescence intensity (F_c/F_m, %) was calculated at every 10 sec and plotted against time. F_c, fluorescence intensity of the cytosol; F_m, fluorescence intensity of the plasma membrane. (Upper panel) PKCβ-II-GFP, initially located in the cytosol, rapidly translocated to the plasma membrane upon Ca²⁺ influx via reverse NCX1. PKCβ-II-GFP returned to the cytosol when NCX1 was turned off. (Lower panel) No significant translocation of PH domain was monitored during the Ca²⁺ transient.

The effects of protein kinase C modulators on rundown. (A) Pretreatment of the cells with a selective PKC inhibitor, calphostin C (1μM), did not inhibit rundown of the Ca²⁺ transients. (B) Pretreatment of a strong PKC activator, PMA (0.1μ M), did not change rundown. (C) Normal rundown was recorded at the beginning of the recording (the first 3 repeats), and the 0.1μM STS was applied to the cells. The post-treatment of staurosporine (STS) recovered the Ca²⁺ transients and the final amplitudes were higher than the initial one. (D) Summarized data are illustrated. Control, rundown from control cells (n=8); Calp-C, pretreatment with calphostin C (n=6); PMA, pretreatment of phorbol ester (n=6); STS, post-treatment of STS after the 3^rd transient (n=4). The black bars in C and D denote the period of time with STS.

Staurosporine (STS) stimulated Na⁺/Ca²⁺ exchanger in a reversible and a dose-dependent manner. (A) In this recording, the initial Ca²⁺ transient was followed by a complete rundown of NCX1 (i.e. the 2^nd transient was completely disappeared). When STS (0.1μM) was briefly applied prior to the 3^rd one, it rapidly restored the Ca²⁺ transients. After the washing of STS, the next rundown was processed. By the second application of STS, restoration of the transients started again. (B) A lower concentration of STS (50 nM), that was applied before the initial stimulation, significantly inhibited rundown (compare the amplitudes of the initial 3 Ca²⁺ transients). When the concentrations of STS were increased up to 0.3μM, the amplitudes of the transients were increased dose-dependently.

STS-increased Na⁺/Ca²⁺ exchanger is independent of protein kinase C and/or protein kinase A. (A~C) Even in the presence of calphostin C (1μM), PMA (0.1μM), and a PKA activator forskolin (FSK, 1μM), rundown of NCX1 was normally developed (i.e. the 2^nd or 3^rd transient was markedly decreased). However, the addition of STS (0.1μM) with each chemical almost completely restored the Ca²⁺ transients. STS was applied after the recording of the 2^nd Ca²⁺ transient. (D) Summarized data were illustrated with time courses of rundown. Calp-C, calphostin C with STS (n=4); PMA, PMA with STS (n=7); FSK, forskolin with STS (n=5).