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Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Pande J, Szewczyk MM, Kuszczak I, Grover S, Escher E, Grover AK - J. Cell. Mol. Med. (2008)

Bottom Line: Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3.We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle.We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON, Canada.

ABSTRACT
Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.

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Effect of caloxin 1c2 on contraction of Na+ loaded coronary artery smooth muscle. (A) Representative trac-ings. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs’ solution and then Na+-loaded by incubation in K+-free Ca2+-free solution for 45 min and then 100 μM L-NAME and 30 μM CPA were added and after another 45 min 20 μM calox-in 1c2 was added to some tissues and equal amount of ethanol to the control tissues. After another 15 min, CaCl2 was added to obtain the specified concentrations of free Ca2+ compensating for the EGTA present previously. Contraction of each artery was expressed as percent of that obtained with 60 mM KCl. The contraction bar indicates a force of contraction 20% of that obtained with 60 mM KCl.(B) A histogram comparing the force of contraction obtained at 50, 100 and 1600 μM Ca2+. The values are mean ± SEM of 11 replicates.
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fig06: Effect of caloxin 1c2 on contraction of Na+ loaded coronary artery smooth muscle. (A) Representative trac-ings. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs’ solution and then Na+-loaded by incubation in K+-free Ca2+-free solution for 45 min and then 100 μM L-NAME and 30 μM CPA were added and after another 45 min 20 μM calox-in 1c2 was added to some tissues and equal amount of ethanol to the control tissues. After another 15 min, CaCl2 was added to obtain the specified concentrations of free Ca2+ compensating for the EGTA present previously. Contraction of each artery was expressed as percent of that obtained with 60 mM KCl. The contraction bar indicates a force of contraction 20% of that obtained with 60 mM KCl.(B) A histogram comparing the force of contraction obtained at 50, 100 and 1600 μM Ca2+. The values are mean ± SEM of 11 replicates.

Mentions: We also examined the effect of caloxin 1c2 on de-endothelialized coronary artery rings, which were Na+-loaded and SERCA was inhibited with CPA. Incubating smooth muscle in K+-free Krebs’ solution Na+ loads the smooth muscle tissues that contract in presence of Ca2+[49, 54]. Depending on the Na+ and Ca2+ electrochemical gradients, NCX can operate in forward mode (Ca2+ extrusion) or in reverse mode (Ca2+ entry). In the Na+-loaded cells, NCX would only allow Ca2+ entry into the cell but not Ca2+ extrusion. Thus, PMCA would be the only system that could lower cytosolic Ca2+ levels. We incubated the tissues in K+-free and Ca2+-free Krebs’ solution, and added CPA to inhibit SERCA. The force of contraction increased in all the tissues when Ca2+ concentration in the medium was increased. However, at 0.05 mM extracellular Ca2+, tissues in caloxin 1c2 showed significantly greater force of contraction (P= 0.019) than the control tissues (Fig. 6). Caloxin 1c2 did not affect the force of contraction at 0.1 (P= 0.2267) and 1.6 (P= 0.4717) mM extracellular Ca2+. Thus, caloxin 1c2 increased Ca2+ sensitivity of the tissues when they were Na+-loaded and SERCA was inhibited. However, the maximum force of contraction was not altered.


Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Pande J, Szewczyk MM, Kuszczak I, Grover S, Escher E, Grover AK - J. Cell. Mol. Med. (2008)

Effect of caloxin 1c2 on contraction of Na+ loaded coronary artery smooth muscle. (A) Representative trac-ings. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs’ solution and then Na+-loaded by incubation in K+-free Ca2+-free solution for 45 min and then 100 μM L-NAME and 30 μM CPA were added and after another 45 min 20 μM calox-in 1c2 was added to some tissues and equal amount of ethanol to the control tissues. After another 15 min, CaCl2 was added to obtain the specified concentrations of free Ca2+ compensating for the EGTA present previously. Contraction of each artery was expressed as percent of that obtained with 60 mM KCl. The contraction bar indicates a force of contraction 20% of that obtained with 60 mM KCl.(B) A histogram comparing the force of contraction obtained at 50, 100 and 1600 μM Ca2+. The values are mean ± SEM of 11 replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401146&req=5

fig06: Effect of caloxin 1c2 on contraction of Na+ loaded coronary artery smooth muscle. (A) Representative trac-ings. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs’ solution and then Na+-loaded by incubation in K+-free Ca2+-free solution for 45 min and then 100 μM L-NAME and 30 μM CPA were added and after another 45 min 20 μM calox-in 1c2 was added to some tissues and equal amount of ethanol to the control tissues. After another 15 min, CaCl2 was added to obtain the specified concentrations of free Ca2+ compensating for the EGTA present previously. Contraction of each artery was expressed as percent of that obtained with 60 mM KCl. The contraction bar indicates a force of contraction 20% of that obtained with 60 mM KCl.(B) A histogram comparing the force of contraction obtained at 50, 100 and 1600 μM Ca2+. The values are mean ± SEM of 11 replicates.
Mentions: We also examined the effect of caloxin 1c2 on de-endothelialized coronary artery rings, which were Na+-loaded and SERCA was inhibited with CPA. Incubating smooth muscle in K+-free Krebs’ solution Na+ loads the smooth muscle tissues that contract in presence of Ca2+[49, 54]. Depending on the Na+ and Ca2+ electrochemical gradients, NCX can operate in forward mode (Ca2+ extrusion) or in reverse mode (Ca2+ entry). In the Na+-loaded cells, NCX would only allow Ca2+ entry into the cell but not Ca2+ extrusion. Thus, PMCA would be the only system that could lower cytosolic Ca2+ levels. We incubated the tissues in K+-free and Ca2+-free Krebs’ solution, and added CPA to inhibit SERCA. The force of contraction increased in all the tissues when Ca2+ concentration in the medium was increased. However, at 0.05 mM extracellular Ca2+, tissues in caloxin 1c2 showed significantly greater force of contraction (P= 0.019) than the control tissues (Fig. 6). Caloxin 1c2 did not affect the force of contraction at 0.1 (P= 0.2267) and 1.6 (P= 0.4717) mM extracellular Ca2+. Thus, caloxin 1c2 increased Ca2+ sensitivity of the tissues when they were Na+-loaded and SERCA was inhibited. However, the maximum force of contraction was not altered.

Bottom Line: Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3.We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle.We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON, Canada.

ABSTRACT
Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.

Show MeSH
Related in: MedlinePlus