Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Abstract

Assays for complete quantification of Na+, K(+)- and Ca(2+)-ATPase in crude homogenates of rat ventricular myocardium by determination of K(+)- and Ca(2+)-dependent p-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K(+)- and Ca(2+)-dependent pNPPase activities in ventricular myocardium of 11-12 week-old rats were found to be 2.98 +/- 0.10 and 0.29 +/- 0.02 mumol x min-1 x g-1 wet wt. (mean +/- SEM) (n = 5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na+, K(+)- and Ca(2+)-ATPase concentrations were estimated to 2 and 10 nmol x g-1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca(2+)-dependent pNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K(+)-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple outcome of variations in water and protein content of myocardium. Expressed per heart, the K(+)- and Ca(2+)-dependent pNPPase activity gradually increased to a plateau. The present assay for Na+, K(+)-ATPase quantification has the advantage over [3H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) in crude tissue homogenates. Furthermore, with few modifications the pNPPase assay allows quantification of Ca(2+)-ATPase on crude myocardial homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Astragaloside IV Improved Intracellular Calcium Handling in Hypoxia-Reoxygenated Cardiomyocytes via the Sarcoplasmic Reticulum Ca²⁺-ATPase

“…2+ -ATPase Activity SR Ca 2+ -ATPase activity was assayed by a modified p -NPP method [19] routinely used in our laboratory [15,20,21] . Cardiomyocytes were homogenized and centrifuged at 4 ° C for 20 min.…”

rAAV‐asPLB transfer attenuates abnormal sarcoplasmic reticulum Ca2+‐ATPase activity and cardiac dysfunction in rats with myocardial infarction

“…SERCA activity was determined following the method of Larsen [17], with some modifications. The myocardium of the left ventricle was homogenized in tissue buffer (20 mmol/L Hepes, 2 mmol/L EDTA and 250 mmol/L sucrose, pH 7.4) at 4°C.…”

“…Unphosphorylated PLB inhibits SERCA2 activity, whereas phosphorylated PLB dissociates from SERCA2 and leads to enhanced pump activity. PLB can be phosphorylated at the serine 16 residue by β-adrenergic signalling and at the threonine 17 residue via calcium/calmodulin kinase II [11]. It has been shown that reduced serine 16 phosphorylation of PLB (Pser16-PLB) in the failing rat myocardium is a major contributor to decreased SERCA2 activity [12].…”

Astragaloside IV Alleviates Hypoxia/Reoxygenation-Induced Neonatal Rat Cardiomyocyte Injury via the Protein Kinase A Pathway

“…SERCA2a activity was measured by a modified p-nitrophenyl phosphate disodium salt hexahydrate (p-NPP; Sigma) method [16]. In brief, cultured cardiomyocytes were homogenized in a buffer containing 20 mmol/l HEPES, 2 mmol/l EDTA and 250 mmol/l sucrose, and centrifuged at 12,000 rpm for 20 min at 4°C.…”

“…PKA is a cAMP-dependent protein kinase which is activated under β-adrenergic stimulation and cAMP level increase. PKA plays a significant role in physiological and pathological conditions by phosphorylating PLN at Ser 16 and reversing its inhibition of SERCA2a [7][8][9]. Astragalus membranaceus (Fisch.)…”