Influence of NO and [Ca²⁺]_o on [Ca²⁺]_i homeostasis in rat ventricular cardiomyocytes

Abstract

In this study, we explored the effect of NO-induced changes in [Ca 2þ ] i homeostasis in rat ventricular cardiomyocytes through variation in extracellular Ca 2þ and application of SNAP (Snitroso-N-acetyl-D,L-penicillamine) as an NO-donor. The Fura-2 signal dynamics and phalloidin intensity profile of z-disks in rat cardiomyocytes served as endpoints to describe the mechanisms involved in the control of the intracellular Ca 2þ levels, depending on the kinetics of distribution of the SNAP-produced NO. The results demonstrated that SNAP caused small phalloidin intensity profile changes between the z-lines in the presence of [Ca 2þ ] o. However, in the absence of [Ca 2þ ] o , SNAP in a concentration of 300 lmol/L induced a significant decrease in the distance between z-lines. This SNAP-induced decrease was reflected as a decrease in the phalloidin fluorescence intensity in the middle of the sarcomere, due to the preferential imaging of greater fluorescence in the bulk of the thin filaments. Based on the Ca 2þ fluorescence intensity profile, we could suggest that intracellular Ca 2þ is mainly affected by the mechanisms of Ca 2þ outflow, rather than the mechanisms of Ca 2þ inflow in the presence of NO. Actually, through variation in the electrochemical gradient of Ca 2þ , we induced mechanisms of faster/slower cytosolic Ca 2þ emptying. The obtained data showed that the delay in the cytosolic Ca 2þ emptying in the presence of [Ca 2þ ] o is due to the increased electrochemical gradient of Ca 2þ .

Cardiomyocytes' prolonged IL‐2 incubation induces enhancement in L‐type Ca2+ channels mediated by inhibitory‐kappaB kinase/nuclear factor‐kappaB signalling

“…Kinetics of IL‐2‐induced intracellular nitric oxide (NO) production were assessed by the incubation of cardiomyocytes with NO‐sensitive fluorescent dye 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM, 5 µmol/L) in 3 mL of standard Kraftbrühe solution for 30 min 20 . Further, cardiomyocytes were washed for 30 min and transferred from suspension to a perfusion chamber where they adhered freely to the glass bottom 21‐24 . 15‐20 min after precipitation, cells were incubated in a standard Kraftbrühe solution containing 2 ng/mL IL‐2.…”

“…20 Further, cardiomyocytes were washed for 30 min and transferred from suspension to a perfusion chamber where they adhered freely to the glass bottom. [21][22][23][24] 15-20 min after precipitation, cells were incubated in a standard Kraftbrühe solution containing 2 ng/mL IL-2. Fluorescence (in time intervals of 1, 5, 10, 30, 60 and 120 min) was measured prior to and after IL-2 incubation of the same cell at λex = 495 and λem = 515 nm, respectively.…”