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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 basal tone of coronary artery. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs'solution and incubated in this solution containing 100 μM L-NAME for 1 hr and then 0, 10, 20 or 50 μM caloxin 1c2 was added. An increase in contraction in 20 min after addition of the caloxin 1c2 was determined as mN/mg tissue weight. The values are mean ± SEM of 18, 12, 7 and 7 replicates for 0, 10, 20 or 50 μM caloxin 1c2, respectively.
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fig05: Effect of caloxin 1c2 on basal tone of coronary artery. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs'solution and incubated in this solution containing 100 μM L-NAME for 1 hr and then 0, 10, 20 or 50 μM caloxin 1c2 was added. An increase in contraction in 20 min after addition of the caloxin 1c2 was determined as mN/mg tissue weight. The values are mean ± SEM of 18, 12, 7 and 7 replicates for 0, 10, 20 or 50 μM caloxin 1c2, respectively.

Mentions: We next examined the effects of 0, 10, 20 and 50 μM caloxin 1c2 on basal tone of de-endothelialized coronary artery [42, 49, 53]. The final ethanol concentration in all the four groups was 0.01%. Time course of the tone development was examined (data not shown). There was no significant effect of caloxin 1c2 within 2 min. Only 20 and 50 μM caloxin 1c2 gave an increase after 5 min, whereas 10 μM caloxin 1c2 also gave a significant increase after 10 min. At all the three caloxin 1c2 concentrations, the caloxin induced increase in basal tone leveled off after 20 min at 2–3% of the maximum response obtained with 60 mM KCl (Fig. 5). A multiple comparison test using one way-ANOVA negated the hypothesis that the differences in contraction in 0, 10, 20 and 50 μM caloxin 1c2 were purely due to chance (P= 0.0015). In a Tukey–Kramer multiple comparison test, q values of greater than 3.807 would indicate that a group differed from another comparison group. In this test there was significant difference between control tissues and those challenged with 10 (q = 4.085, P < 0.05), 20 (q = 5.235, P < 0.05) and 50 (q = 4.156, P < 0.05) μM caloxin 1c2. Furthermore, there was no significant difference between tissues treated with 10 or 20 (q = 1.435, P > 0.05), 10 or 50 (q = 0.7679, P > 0.05) and 20 or 50 (q = 0.5133, P > 0.05) μM caloxin 1c2.


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 basal tone of coronary artery. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs'solution and incubated in this solution containing 100 μM L-NAME for 1 hr and then 0, 10, 20 or 50 μM caloxin 1c2 was added. An increase in contraction in 20 min after addition of the caloxin 1c2 was determined as mN/mg tissue weight. The values are mean ± SEM of 18, 12, 7 and 7 replicates for 0, 10, 20 or 50 μM caloxin 1c2, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401146&req=5

fig05: Effect of caloxin 1c2 on basal tone of coronary artery. De-endothelialized coronary artery rings were contracted in 60 mM KCl, washed in Krebs'solution and incubated in this solution containing 100 μM L-NAME for 1 hr and then 0, 10, 20 or 50 μM caloxin 1c2 was added. An increase in contraction in 20 min after addition of the caloxin 1c2 was determined as mN/mg tissue weight. The values are mean ± SEM of 18, 12, 7 and 7 replicates for 0, 10, 20 or 50 μM caloxin 1c2, respectively.
Mentions: We next examined the effects of 0, 10, 20 and 50 μM caloxin 1c2 on basal tone of de-endothelialized coronary artery [42, 49, 53]. The final ethanol concentration in all the four groups was 0.01%. Time course of the tone development was examined (data not shown). There was no significant effect of caloxin 1c2 within 2 min. Only 20 and 50 μM caloxin 1c2 gave an increase after 5 min, whereas 10 μM caloxin 1c2 also gave a significant increase after 10 min. At all the three caloxin 1c2 concentrations, the caloxin induced increase in basal tone leveled off after 20 min at 2–3% of the maximum response obtained with 60 mM KCl (Fig. 5). A multiple comparison test using one way-ANOVA negated the hypothesis that the differences in contraction in 0, 10, 20 and 50 μM caloxin 1c2 were purely due to chance (P= 0.0015). In a Tukey–Kramer multiple comparison test, q values of greater than 3.807 would indicate that a group differed from another comparison group. In this test there was significant difference between control tissues and those challenged with 10 (q = 4.085, P < 0.05), 20 (q = 5.235, P < 0.05) and 50 (q = 4.156, P < 0.05) μM caloxin 1c2. Furthermore, there was no significant difference between tissues treated with 10 or 20 (q = 1.435, P > 0.05), 10 or 50 (q = 0.7679, P > 0.05) and 20 or 50 (q = 0.5133, P > 0.05) μM caloxin 1c2.

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