A new generation of Ca2+ indicators with greatly improved fluorescence properties.

Microprocessor-controlled changes of [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils of a skinned canine cardiac Purkinje cell and aequorin bioluminescence recording were used to study the mechanism of Ca2+-induced release of Ca2+ from the SR. This Ca2+ release is triggered by a rapid increase of [free Ca2+] at the outer surface of the SR of a previously quiescent skinned cell. Ca2+-induced release of Ca2+ occurred under conditions that prevented any synthesis of ATP from ADP, was affected differentially by interventions that depressed the SR Ca2+ pump about equally, and required ionic conditions incompatible with all known Ca2+-releasing, uncoupled, partial reactions of the Ca2+ pump. Increasing the [free Ca2+]trigger up to an optimum increased the amount of Ca2+ released. A supraoptimum increase of [free Ca2+] trigger inactivated Ca2+-induced release of Ca2+, but partial inactivation was also observed at [free Ca2+] below that necessary for its activation. The amplitude of the Ca2+ release induced by a given increase of [free Ca2+] decreased when the rate of this increase was diminished. These results suggest that Ca2+-induced release of Ca2+ is through a channel across the SR membrane with time- and Ca2+-dependent activation and inactivation. The inactivating binding site would have a higher affinity for Ca2+ but a lower rate constant than the activating site. Inactivation appeared to be a first-order kinetic reaction of Ca2+ binding to a single site at the outer face of the SR with a Q10 of 1.68. The removal of inactivation was the slowest step of the cycle, responsible for a highly temperature-dependent (Q10 approximately 4.00) refractory period.

https://rupress.org/jgp/article-pdf/85/2/247/1248727/247.pdf

Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell.

�Microprocessor-controlled changes of [free Ca 2 '] at the outer surface of the Sarcoplasmic reticulum (SR) wrapped around individual myofibrils of a skinned canine cardiac Purkinje cell and aequorin bioluminescence recording were used to study the mechanism of Ca 2 '-induced release of Ca 21 from the SR . This Ca2' release is triggered by a rapid increase of [free Ca 21] at the outersurface of the SR of a previously quiescent skinned cell . Ca 2 '-induced

Calcium-induced Release of Calcium from the Sarcoplasmic Reticulum of a Skinned Canine Cardiac Purkinje Cell

To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

https://www.physiology.org/doi/pdf/10.1152/physrev.1999.79.4.1089

Measurement of Intracellular Calcium

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

Re-Examination of the Apparent Binding Constant of Ethylene Glycol Bis(β-Aminoethyl Ether)-N,N,N′N′-Tetraacetic Acid with Calcium around Neutral pH

1. The characteristics of four calcium indicators, murexide(MX), tetramethylmurexide(TMX), arsenazo III (Az) and antipyrylazo III (Ap), were examined to assess their applicability for biological experiments. 2. Az has the following serious disadvantages, although it has a high sensitivity to Ca: (i) nonlinearity of absorbance change against the concentration of Ca due to a change in the structural composition of its complex (2 : 1 complex to 1 : 1 complex) in the range below 10 micro M Ca2+, and due to the high apparent binding constant to Ca at 20 micro M or higher Ca2+; (ii) severe interference by Mg2+; (iii) rather low association and dissociation rate constants with Ca which are not sufficient under conditions where Ca2+ to be determined in the system cannot be disturbed by Az; (iv) inhibitory effect on Ca uptake by bullfrog fragmented sarcoplasmic reticulum. 3. Ap has similar disadvantages, although Ap reacts with Ca faster than Az. (i) Ap suffers from interference by Mg2+ even though the effect of Mg2+ can be minimized at the expense of reduced sensitivity. (ii) Plots of absorbance change versus the concentration of Ca deviate from linearity around 20 micro M Ca2+ or higher. (iii) Ap is not very sensitive at concentrations which would not disturb Ca2+ in the system to be determined. TMX is twice as sensitive as MX. However, TMX may be more permeable through membranes than MX in the presence of ATP. MX has no disadvantages except for its low sensitivity to Ca2+.

Comparison of the characteristics of four metallochromic dyes as potential calcium indicators for biological experiments.

While many reports have shown that Ca2+ alone causes ryanodine binding to the heavy fraction of the sarcoplasmic reticulum (HFSR), our results demonstrate that caffeine or beta,gamma-methylene adenosine triphosphate (AMPOPCP) in addition to Ca2+ is necessary for ryanodine binding, although Ca2+ is indispensable for it. While clarifying the reasons for this discrepancy, we found that a high osmolarity of the reaction medium, but not ionic strength, is a crucial factor. In a hypertonic solution containing 1 M NaCl, Ca2+ alone causes a sizable extent of ryanodine binding. Caffeine and AMPOPCP independently stimulate it, unlike the case of 0.17 M KCl (or NaCl) medium, in which they show a potentiating interaction. Ryanodine binding in the hypertonic solution was markedly enhanced not only as to the binding rate but also the extent. The Scatchard plot was linear, indicating a single class of homogeneous binding sites. The maximum number of binding sites as well as the affinity was also increased in 1 M NaCl-medium. The presence of AMPOPCP and/or caffeine did not affect the magnitudes of them so much, especially that of the affinity, in the hypertonic medium, as in the isotonic medium. The Ca2(+)-dependence of ryanodine binding in the stimulatory range was similar to that in 0.17 M KCl- (or NaCl-) medium. However, the very weak inhibition at high Ca2+ concentrations is in striking contrast to ryanodine binding in the isotonic medium. The stimulation due to a high osmolarity is distinct, as to the mechanism, from that due to AMPOPCP, caffeine, or temperature. The dissociation of [3H]ryanodine bound was also examined under various experimental conditions.

Osmolarity-Dependent Characteristics of [3H]Ryanodine Binding to Sarcoplasmic Reticulum

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle.

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.

Hikaru Harafuji

Papers

Re-Examination of the Apparent Binding Constant of Ethylene Glycol Bis(β-Aminoethyl Ether)-N,N,N′N′-Tetraacetic Acid with Calcium around Neutral pH

Comparison of the characteristics of four metallochromic dyes as potential calcium indicators for biological experiments.

Osmolarity-Dependent Characteristics of [3H]Ryanodine Binding to Sarcoplasmic Reticulum

Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle.

Ca-release by phosphoinositides from sarcoplasmic reticulum of frog skeletal muscle.