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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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NCX1 and phospholemman expression in EC and SMC. A. RT-PCR using NCX1, G3PDH (glyceraldehyde phosphate dehydrogenase) and PLEM (phospholemman) specific primers. B. Western blots with antibody R3F1 showing NCX1 protein abundance.
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fig04: NCX1 and phospholemman expression in EC and SMC. A. RT-PCR using NCX1, G3PDH (glyceraldehyde phosphate dehydrogenase) and PLEM (phospholemman) specific primers. B. Western blots with antibody R3F1 showing NCX1 protein abundance.

Mentions: NCX1 contains six cryptic exons that can result in alternative splicing [21, 25, 33, 34]. Primers flanking the cryptic exons were used to determine the NCX1 iso-forms expressed in EC and SMC (Table 1). One major (309 bp) and a slightly larger minor band was observed in RT-PCR products of EC and SMC RNA (Fig. 4A).The two bands were sequenced. Translation of the sequences showed that the 309-bp band corresponded to NCX1.3 and the larger one to NCX1.7. A PM protein, phospholemman, inhibits NCX1 [42].Therefore, we also determined if EC and SMC contain mRNA for this protein. RT-PCR product for phospholemman mRNA (277 bp) was detected in SMC but not in EC. The identity of this band was confirmed by sequencing.


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)

NCX1 and phospholemman expression in EC and SMC. A. RT-PCR using NCX1, G3PDH (glyceraldehyde phosphate dehydrogenase) and PLEM (phospholemman) specific primers. B. Western blots with antibody R3F1 showing NCX1 protein abundance.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: NCX1 and phospholemman expression in EC and SMC. A. RT-PCR using NCX1, G3PDH (glyceraldehyde phosphate dehydrogenase) and PLEM (phospholemman) specific primers. B. Western blots with antibody R3F1 showing NCX1 protein abundance.
Mentions: NCX1 contains six cryptic exons that can result in alternative splicing [21, 25, 33, 34]. Primers flanking the cryptic exons were used to determine the NCX1 iso-forms expressed in EC and SMC (Table 1). One major (309 bp) and a slightly larger minor band was observed in RT-PCR products of EC and SMC RNA (Fig. 4A).The two bands were sequenced. Translation of the sequences showed that the 309-bp band corresponded to NCX1.3 and the larger one to NCX1.7. A PM protein, phospholemman, inhibits NCX1 [42].Therefore, we also determined if EC and SMC contain mRNA for this protein. RT-PCR product for phospholemman mRNA (277 bp) was detected in SMC but not in EC. The identity of this band was confirmed by sequencing.

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