Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples

Abstract

Introduction In skeletal muscle, the Na+/K+ ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5–10 mg) and in human skeletal muscle. Methods Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca2+ ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize “background noise.” Results We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized “background noise” (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. Conclusion The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Resting metabolic rate and skeletal muscle SERCA and Na + /K + ATPase activities are not affected by fish oil supplementation in healthy older adults

“…Two to four resting muscle biopsies (total ~50 to 150 mg) were obtained under local anesthesia (2% lidocaine without epinephrine) from the vastus lateralis muscle, using the percutaneous needle biopsy technique described by Bergstrom (1975). A portion of muscle (~50 mg) was immediately diluted 11:1 (vol/wt) in ice‐cold homogenizing buffer containing (in mM) 0.2 PMSF, 250 sucrose, 5 HEPES, and 0.2% NaN3 (pH 7.5), using a hand‐held glass homogenizer (Jannas‐Vela et al, 2019). The homogenate was separated into 2–5 aliquots, flash‐frozen in liquid nitrogen, and stored at −80°C for future analyses of ATPase activities and western blotting of selected proteins.…”

“…Total SERCA activity was measured using a fluorometric assay (Jannas‐Vela et al, 2019). The reaction buffer contained (in mM) 200 KCl, 20 HEPES, 15 MgCl 2 , 1 EGTA, 10 NaN 3 , 5 ATP, and 10 phospho(enol)pyruvate (pH 7.0 at 37°C).…”

“…To estimate SERCA permeability, an ionophore ratio was calculated in which maximal SERCA activity with ionophore was divided by the maximal SERCA activity without ionophore (Jannas‐Vela et al, 2019). One participant from the OO group was excluded due to an inability to accurately measure SERCA activity.…”

“…Measurement of NKA activity was performed with an enzyme‐coupled method using an ATP regenerating assay (Jannas‐Vela et al, 2019). After freeze‐thawing muscle homogenates 5 times, an aliquot (10 μl) was incubated at 37°C in a buffer containing (in mM) 40 Tris HCl, 15 KCl, 1 EGTA, 5 MgCl 2 , 1.5 phospho(enol)pyruvate, 3 ATP (pH 7.4), in the presence of 18 U/ml of PK and LDH, 25 μM of blebbistatin, and 0.3 mM NADH.…”

Повышенный Уровень Креатинфосфокиназы У Больных С Отдаленными Последствиями После Перенесенной Закрытой Черепно-Мозговой Травмы

“…Оригінальні дослідження /Original Researches/ is typical for central nervous system injury and dysfunction resulting in a reversal of glutamate uptake and neuronal depolarization through various excitotoxic neurobiochemical mechanisms and these mechanisms depend on both energy dysfunction in neurons and primary brain injury severity [7][8][9]12]. Inflammatory cells also mediate this secondary brain injury via the release of many pro-inflammatory cytokines that contribute to activation of cell-death cascades or postsynaptic receptor modifications [18,19].…”