Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms

1. The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can he disrupted by the immunosuppressant PK506. We have investigated the effect of FK508 on Ca2+ release and Ca2+ uptake in permeabilized cell types. 2. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC(3)H1 skeletal muscle cells. Ca-45(2+) fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. 3. Addition of PK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to similar to 30% of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. 4. This FK606-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated Ca-45(2+) uptake in SH-ST5Y microsomes was inhibited by FR506 with an IC50 of 19 mu M. 5. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. 6. FK506 (10 mu M) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC(3)H1 cells. 7. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.