The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional

Bartolommei, Gianluca; Tadini‐Buoninsegni, Francesco; Moncelli, Maria Rosa; Gemma, Sandra; Camodeca, Caterina; Butini, Stefania; Campiani, Giuseppe; Lewis, David E.; Inesi, Giuseppe

doi:10.1074/jbc.m111.287276

Cited by 12 publications

(11 citation statements)

References 45 publications

(35 reference statements)

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“…Color fully develops in 30 minutes and solutions have a stability of about 4 hours [23]. We used the Lanzetta method in our previous studies, confirming all these experimental findings [30], [38], [39].…”

Section: Discussionsupporting

confidence: 80%

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A Method to Measure Hydrolytic Activity of Adenosinetriphosphatases (ATPases)

2013

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The detection of small amounts (nanomoles) of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases), that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate (originally employed for phosphate detection in environmental analysis) to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

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Section: Discussionsupporting

confidence: 80%

“…The detection of small amounts (nanomoles) of inorganic phosphate has a broad interest that spans from environmental to biochemical fields [6], [7], [17], [18], [23], [30]–[32]. The present method optimizes an experimental protocol usually employed for environmental analysis to allow its use with phosphatase enzymes, such as ATPases.…”

Section: Discussionmentioning

confidence: 99%

A Method to Measure Hydrolytic Activity of Adenosinetriphosphatases (ATPases)

2013

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“…To confirm that the stimulatory effect of istaroxime on SERCA2a activity translates into a functional effect on Ca 2+ movement, we applied an experimental method to measure charge transfer within the first catalytic cycle of SERCA2a across the SR membrane (Tadini‐Buoninsegni et al ., 2006; 2008b; Bartolommei et al ., ; Lewis et al ., ). Native SR SERCA2a from dog healthy hearts was characterized by pre‐steady state charge measurements on an SSM.…”

Section: Resultsmentioning

confidence: 98%

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Ferrandi

Barassi

Tadini‐Buoninsegni

et al. 2013

British J Pharmacology

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BACKGROUND AND PURPOSECalcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca 2+ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. EXPERIMENTAL APPROACHTo study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. KEY RESULTSWe showed that istaroxime enhances SERCA2a activity, Ca 2+ uptake and the Ca 2+ -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca 2+ cycling. CONCLUSIONS AND IMPLICATIONSIstaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca 2+ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way. LINKED ARTICLE AbbreviationsAHF, acute heart failure; CPA, cyclopiazonic acid; +dP/dt, rate of LV pressure rise; −dP/dt, rate of LV pressure decay; LV, left ventricle; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca 2+

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“…3 ). The charge obtained by numerical integration of the ATP-induced current transient is due to an electrogenic event corresponding to the translocation and release of bound Ca 2+ upon phosphorylation by ATP 35 36 41 within the first enzyme cycle.…”

Section: Resultsmentioning

confidence: 99%

Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

Tadini‐Buoninsegni

Moncelli

Peruzzi

et al. 2015

Sci Rep

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The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

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The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional

Cited by 12 publications

References 45 publications

A Method to Measure Hydrolytic Activity of Adenosinetriphosphatases (ATPases)

A Method to Measure Hydrolytic Activity of Adenosinetriphosphatases (ATPases)

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

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