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Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle.

Szewczyk MM, Davis KA, Samson SE, Simpson F, Rangachari PK, Grover AK - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation.Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots.PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.

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PMCA activity and protein abundance in PM-enriched fractions of EC and SMC. A. Western blot showing relative protein abundance with the anti-body 5F10 (recognizes all PMCA isoforms). B. A plot of pixel volumes (intensity × area) versus proteinamount for bands shown in A. Linear regression gave a slope of 21.7 ± 1.6 for SMC and 4.3 ± 0.2 for EC. The linear correlation coefficients for SMC and EC were 0.9820 and 0.9944, respectively. C. Relative values of PMCA activity and protein abundance with 5F10 (total PMCA), anti-PMCA4 and anti-PMCA1 in Western blots. The relative value for each parameter was computed taking the mean value in SMC as 100%.
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fig02: PMCA activity and protein abundance in PM-enriched fractions of EC and SMC. A. Western blot showing relative protein abundance with the anti-body 5F10 (recognizes all PMCA isoforms). B. A plot of pixel volumes (intensity × area) versus proteinamount for bands shown in A. Linear regression gave a slope of 21.7 ± 1.6 for SMC and 4.3 ± 0.2 for EC. The linear correlation coefficients for SMC and EC were 0.9820 and 0.9944, respectively. C. Relative values of PMCA activity and protein abundance with 5F10 (total PMCA), anti-PMCA4 and anti-PMCA1 in Western blots. The relative value for each parameter was computed taking the mean value in SMC as 100%.

Mentions: The relative abundance of PMCA proteins was determined using three different antibodies: one that recognized all PMCA isoforms (5F10), anti-PMCA4 (JA9) and a polyclonal anti-PMCA1 antibody. Figure 2A shows the results of the Western blot using 5F10 in one experiment. Both SMC and EC gave a band at 140 kDa corresponding to PMCA. Thus, the PMCA protein abundance based on Figure 2B in EC was 22 ±2% of that in SMC. In similar experiments using the anti-PMCA1 antibody, the abundance in EC was 27 ±4% of that in SMC. In contrast, PMCA4 in EC was much smaller (8 ±1% of the SMC value). These results are consistent with those of the PCR experiments in Fig. 1 in that EC express mainly PMCA1.


Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle.

Szewczyk MM, Davis KA, Samson SE, Simpson F, Rangachari PK, Grover AK - J. Cell. Mol. Med. (2007 Jan-Feb)

PMCA activity and protein abundance in PM-enriched fractions of EC and SMC. A. Western blot showing relative protein abundance with the anti-body 5F10 (recognizes all PMCA isoforms). B. A plot of pixel volumes (intensity × area) versus proteinamount for bands shown in A. Linear regression gave a slope of 21.7 ± 1.6 for SMC and 4.3 ± 0.2 for EC. The linear correlation coefficients for SMC and EC were 0.9820 and 0.9944, respectively. C. Relative values of PMCA activity and protein abundance with 5F10 (total PMCA), anti-PMCA4 and anti-PMCA1 in Western blots. The relative value for each parameter was computed taking the mean value in SMC as 100%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401226&req=5

fig02: PMCA activity and protein abundance in PM-enriched fractions of EC and SMC. A. Western blot showing relative protein abundance with the anti-body 5F10 (recognizes all PMCA isoforms). B. A plot of pixel volumes (intensity × area) versus proteinamount for bands shown in A. Linear regression gave a slope of 21.7 ± 1.6 for SMC and 4.3 ± 0.2 for EC. The linear correlation coefficients for SMC and EC were 0.9820 and 0.9944, respectively. C. Relative values of PMCA activity and protein abundance with 5F10 (total PMCA), anti-PMCA4 and anti-PMCA1 in Western blots. The relative value for each parameter was computed taking the mean value in SMC as 100%.
Mentions: The relative abundance of PMCA proteins was determined using three different antibodies: one that recognized all PMCA isoforms (5F10), anti-PMCA4 (JA9) and a polyclonal anti-PMCA1 antibody. Figure 2A shows the results of the Western blot using 5F10 in one experiment. Both SMC and EC gave a band at 140 kDa corresponding to PMCA. Thus, the PMCA protein abundance based on Figure 2B in EC was 22 ±2% of that in SMC. In similar experiments using the anti-PMCA1 antibody, the abundance in EC was 27 ±4% of that in SMC. In contrast, PMCA4 in EC was much smaller (8 ±1% of the SMC value). These results are consistent with those of the PCR experiments in Fig. 1 in that EC express mainly PMCA1.

Bottom Line: Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation.Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots.PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.

Show MeSH
Related in: MedlinePlus