Effects of cytosolic ATP on spontaneous and triggered Ca2+‐induced Ca2+ release in permeabilised rat ventricular myocytes

The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin‐permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura‐2 or Fluo‐3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200–300 nM, in solutions weakly Ca2+ buffered with 0.05 mm EGTA. Alternatively, Ca2+‐induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr‐5 (0.08 mm), replacing EGTA in the solution. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mm to 0.1 mm ATP, reduced the release frequency by 48.6 ± 7 % (n= 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 μM, consistent with inhibition of the SR Ca2+ pump. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. CICR triggered by flash photolysis of Nitr‐5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mm ATP. In the presence of 0.1‐0.2 mm ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release ‘alternans’, whereby every alternate trigger induced a response. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mm to 100 μM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr‐5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia is considered.

[1]  A. Fabiato,et al.  Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell , 1985, The Journal of general physiology.

[2]  A. Duke,et al.  Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin‐permeabilized frog skeletal muscle fibres , 1998, The Journal of physiology.

[3]  Alain Lacampagne,et al.  Modulation of the Frequency of Spontaneous Sarcoplasmic Reticulum Ca2+ Release Events (Ca2+ Sparks) by Myoplasmic [Mg2+] in Frog Skeletal Muscle , 1998, The Journal of general physiology.

[4]  M. Morad,et al.  Ca2(+)-induced Ca2+ release as examined by photolysis of caged Ca2+ in single ventricular myocytes. , 1990, The American journal of physiology.

[5]  Godfrey L. Smith,et al.  Effects of creatine phosphate and inorganic phosphate on the sarcoplasmic reticulum of saponin‐treated rat heart. , 1995, The Journal of physiology.

[6]  Donald M. Bers,et al.  Excitation-Contraction Coupling and Cardiac Contractile Force , 2001, Developments in Cardiovascular Medicine.

[7]  W. Wier,et al.  Mechanism of release of calcium from sarcoplasmic reticulum of guinea‐pig cardiac cells. , 1988, The Journal of physiology.

[8]  L. Xu,et al.  Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions. , 1996, Circulation research.

[9]  D. Eisner,et al.  The effect of tetracaine on supontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes , 1997 .

[10]  G. Smith,et al.  EGTA purity and the buffering of calcium ions in physiological solutions. , 1984, The American journal of physiology.

[11]  J. Kentish,et al.  Effects of changes of pH on the contractile function of cardiac muscle. , 1990, The American journal of physiology.

[12]  D. Allen,et al.  Myocardial contractile function during ischemia and hypoxia. , 1987, Circulation research.

[13]  A. Gurney.  Flash photolysis of caged compounds , 1999 .

[14]  G. Smith,et al.  Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent ions including calcium. , 1985, Biochimica et biophysica acta.

[15]  G. Meissner,et al.  Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. , 1987, The Journal of biological chemistry.

[16]  A. Fabiato.  Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell , 1985, The Journal of general physiology.

[17]  E Niggli,et al.  Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks. , 1999, Annual review of physiology.

[18]  Godfrey L. Smith,et al.  Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles , 1988, The Journal of general physiology.

[19]  Gerhard Meissner,et al.  Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel. , 1986, Biophysical journal.

[20]  D. Eisner,et al.  The effect of tetracaine on spontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes. , 1997, The Journal of physiology.

[21]  L. Xu,et al.  Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel , 1993, The Journal of general physiology.

[22]  A. Fabiato,et al.  Contractions induced by a calcium‐triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. , 1975, The Journal of physiology.

[23]  M. Kawai,et al.  Cs+ inhibits spontaneous Ca2+ release from sarcoplasmic reticulum of skinned cardiac myocytes. , 1998, American journal of physiology. Heart and circulatory physiology.

[24]  A. Duke,et al.  Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres , 1998, Pflügers Archiv.

[25]  W. Lederer,et al.  Calcium sparks and [Ca2+]i waves in cardiac myocytes. , 1996, The American journal of physiology.

[26]  D. Steele,et al.  Inorganic phosphate decreases the Ca2+ content of the sarcoplasmic reticulum in saponin‐treated rat cardiac trabeculae. , 1992, The Journal of physiology.

[27]  J. Kentish,et al.  Effects of inorganic phosphate and ADP on calcium handling by the sarcoplasmic reticulum in rat skinned cardiac muscles. , 1995, Cardiovascular research.

[28]  W. Lederer,et al.  The role of ATP in energy-deprivation contractures in unloaded rat ventricular myocytes. , 1990, Canadian journal of physiology and pharmacology.

[29]  M. Morad,et al.  Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. , 1989, Science.

[30]  A. Fabiato,et al.  Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. , 1979, Journal de physiologie.

[31]  W Yuan,et al.  Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. , 1995, The American journal of physiology.

[32]  J. Hancox,et al.  ”Voltage-activated Ca release” in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP , 1997, Pflügers Archiv.