Calibration of indo-1 and resting intracellular [Ca]i in intact rabbit cardiac myocytes

Abstract

Fluorescent Ca indicators have been extremely valuable in understanding intracellular [Ca] ([Ca]i) regulation in many cell types. The calibration of these indicators in the intracellular environment, however, has been a continuous challenge. We performed in vivo calibrations of indo-1 in isolated rabbit ventricular myocytes loaded with the acetoxymethylester form of indo-1 and used the perforated patch variation of whole cell voltage clamp. Voltage, [Na], and [K] gradients were eliminated to approach equilibrium. We also took advantage of the powerful Na/Ca exchange in cardiac myocytes so that [Ca]i would be equilibrated with [Ca]o (because there was no [Na] or voltage gradient). The equilibration of [Na] and [Ca] across the membrane was tested by measuring the reversal potential of Na current and poking the cell to test for changes in [Ca]i-dependent fluorescence ratio. The apparent dissociation constant, Kd for indo-1 in the cellular environment was 844 nM, which is approximately 2-3 times higher than that in aqueous solutions. In a separate series of experiments, a null point approach was used to determine the [Ca]i in intact cells at rest for very long periods (82 +/- 6 nM). This is lower than that measured 15 s after a train of steady-state twitches ([Ca]i = 294 +/- 53 nM). These experiments also allowed the direct assessment of the shortening versus [Ca]i relationship in intact cells.

β-Adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca 2+ channel

“…Cytosolic [Ca 2ϩ ] was determined with the formula [Ca 2ϩ ] ϭ ␤kd(R Ϫ Rmin)/(Rmax Ϫ R), where ␤kd is the apparent dissociation constant, R ϭ F 405/F485, and Rmin and Rmax represent the R at zero and saturated Ca 2ϩ , respectively. The values of ␤ ⅐ kd, Rmin, and Rmax were calibrated following a published procedure (38).…”

Differential Integration of Ca2+-Calmodulin Signal in Intact Ventricular Myocytes at Low and High Affinity Ca2+-Calmodulin Targets

“…Myocyte Isolation and Adenoviral Infection-Adult rabbit ventricular myocytes were isolated as previously described (31). All of the procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Loyola University Chicago Institutional Animal Care and Use Committee.…”

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart

“…Changes were seen at all excitation wavelengths studied in association with manipulation of NO but the fluorescence changes with 480nm and 490nm were much larger than with 470nm or 500nm. The difference from in vitro data may be due to a number of factors especially considering the different biological conditions and optical path in the whole heart as compared to in vitro cuvette experiments (Bassani et al, 1995). Hence, excitation wavelengths of 480 nm and 490 nm are most suitable for measuring NO activity in the isolated heart using DAF-2.…”

“…This is technically challenging and requires the quenching of fluorescence to obtain minimum fluorescence and increasing the fluorescence to a known maximum concentration of NO. Even in more established fluorescence measurement methods such as Ca 2+ transients, calibrating the intracellular concentration from fluorescence measurements in the whole heart poses significant difficulties and is subject to a multitude of confounding factors and errors (Bassani et al, 1995).…”