Cytosolic energy reserves determine the effect of glycolytic sugar phosphates on sarcoplasmic reticulum Ca2+ release in cat ventricular myocytes

摘要

Localization of glycolytic enzymes in close proximity to Ca²⁺ transport systems of the sarcoplasmic reticulum (SR) in cardiac cells suggests an important functional role for glycolysis in intracellular [Ca²⁺] regulation and, consequently, excitation–contraction coupling. Here, we investigated the mechanisms of regulation of SR Ca²⁺ release by glycolytic sugar phosphate intermediates in cat ventricular myocytes. Experiments with permeabilized myocytes revealed that with normal cytosolic energy reserves (mm: ATP 5, ADP 0.01, phosphocreatine (CrP) 10) fructose-1,6-bisphosphate (FBP; 1 mm) and fructose-6-phosphate (F6P; 1 mm) caused a transient increase of Ca²⁺ spark frequency by 62 and 42%, respectively. This effect of sugar phosphates was associated with a 13% decrease in SR Ca²⁺ load. Pretreatment of the cells with an inhibitor of glycolysis, iodoacetate (IAA; 0.5 mm), did not prevent the effects of FBP and F6P on Ca²⁺ sparks. Recording of single ryanodine receptor (RyR) channel activity indicated that FBP and F6P significantly increased RyR open probability. Reduction of cytosolic energy reserves decreased Ca²⁺ spark activity. Increasing [ADP] to 0.4 mm or removal of CrP ([ATP] was kept constant) caused a slowly developing decrease of Ca²⁺ spark frequency by 29 and 42%, respectively. Changing [ADP] and [CrP] simultaneously decreased Ca²⁺ spark frequency by 66%. This inhibition of Ca²⁺ sparks was associated with a 40% decrease in SR Ca²⁺ load. The subsequent addition of FBP (1 mm) partially restored Ca²⁺ spark frequency and SR Ca²⁺ load. This recovery of Ca²⁺ sparks was blocked completely by IAA. These data suggest that at physiological ATP, ADP and CrP levels accumulation of sugar phosphates from glycolysis can stimulate SR Ca²⁺ release. This effect does not require the activity of downstream glycolytic enzymes, but rather is the result of direct activation of RyRs. However, under conditions associated with depletion of cellular energy reserves (e.g. myocardial ischaemia), ATP generated from glycolysis may play an important role in maintaining myocardial Ca²⁺ homeostasis by improving SR Ca²⁺ uptake.