Untitled

Abstract

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.

Determination of Free Calcium and Calcium-Containing Species in Human Plasma by Capillary Electrophoresis-Inductively Coupled Plasma Optical Emission Spectrometry

“…13 Moreover, the indicators also bind to proteins, and thus, their fluorescent properties might be altered. 14 Similarly, polyvalent cations can interfere with both liquid membrane and solid-state Ca 2+ -selective electrodes. For example, significant interference from Pb 2+ , Fe 2+ , and Hg 2+ on liquid membrane Ca 2+ -selective electrodes were reported when these ions were at concentrations approximately 100-, 0.5-, and 0.25-fold relative to the free Ca 2+ , respectively.…”

“…The fluorescent indicators are not specific to only free Ca 2+ and can be easily affected by coexisting heavy-metal ions (e.g., Mn 2+ , Co 2+ , Zn 2+ , Cd 2+ , Sr 2+ , and Ba 2+ ) that are present in the sample . Moreover, the indicators also bind to proteins, and thus, their fluorescent properties might be altered . Similarly, polyvalent cations can interfere with both liquid membrane and solid-state Ca 2+ -selective electrodes.…”

Identification and analysis of calcium speciation in red blood cells by ultrasonic-dialysis capillary electrophoresis inductively coupled plasma optical emission spectrometry

“…The fluorescent indicators bind not only to free Ca 2+ , but also to proteins, which may then alter their fluorescent properties. 19 In addition, the results can be easily interfered by co-existing heavy-metal ions (e.g., Mn 2+ , Co 2+ , Zn 2+ , Cd 2+ , Sr 2+ , and Ba 2+ ) in the sample. 20 Hyphenated techniques have been applied to speciation analysis.…”