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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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Related in: MedlinePlus

PMCA expression in pig coronary artery SMC and EC. PCR was carried out using PMCA1 and PMCA4 specific primers as indicated or as co-PCR using both sets of primers. Two different dilutions of reverse transcripts were used for co-PCR of each sample.
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fig01: PMCA expression in pig coronary artery SMC and EC. PCR was carried out using PMCA1 and PMCA4 specific primers as indicated or as co-PCR using both sets of primers. Two different dilutions of reverse transcripts were used for co-PCR of each sample.

Mentions: RT-PCR was used to determine the PMCA genes expressed in EC and SMC (Fig. 1). All the primers flanked the cryptic splice site C that lies within PMCA regulatory domain [39]. Sequences of the primers are given in Table 1. The heart and brain cDNA was used to optimize PCR conditions. PCR products for PMCA2 and 3 were not observed in EC or SMC. Expected sizes for PMCA1b, PMCA4a and PMCA4b were 429, 902 and 727 bp, respectively. Although RT-PCR using RNA from EC gave bands for both PMCA1b and 4b, in co-PCR experiments PMCA1b band dominated. In contrast, bands for both PMCA1 and PMCA4 were observed in co-PCR experiments using different dilutions of the reverse transcripts prepared from SMC (Fig. 1). These results are consistent with those previously reported using freshly iso-lated EC and SMC from pig aorta [7, 31]. Although not shown, the following controls gave no PCR products: no reverse transcriptase and no cDNA template. Identities of PMCA1b and PMCA4b bands were also confirmed by the sequencing of gel-purified PCR products. Specificity of the PMCA4a was confirmed by RT-PCR with PMCA4a specific primers (Table 1).


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 expression in pig coronary artery SMC and EC. PCR was carried out using PMCA1 and PMCA4 specific primers as indicated or as co-PCR using both sets of primers. Two different dilutions of reverse transcripts were used for co-PCR of each sample.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: PMCA expression in pig coronary artery SMC and EC. PCR was carried out using PMCA1 and PMCA4 specific primers as indicated or as co-PCR using both sets of primers. Two different dilutions of reverse transcripts were used for co-PCR of each sample.
Mentions: RT-PCR was used to determine the PMCA genes expressed in EC and SMC (Fig. 1). All the primers flanked the cryptic splice site C that lies within PMCA regulatory domain [39]. Sequences of the primers are given in Table 1. The heart and brain cDNA was used to optimize PCR conditions. PCR products for PMCA2 and 3 were not observed in EC or SMC. Expected sizes for PMCA1b, PMCA4a and PMCA4b were 429, 902 and 727 bp, respectively. Although RT-PCR using RNA from EC gave bands for both PMCA1b and 4b, in co-PCR experiments PMCA1b band dominated. In contrast, bands for both PMCA1 and PMCA4 were observed in co-PCR experiments using different dilutions of the reverse transcripts prepared from SMC (Fig. 1). These results are consistent with those previously reported using freshly iso-lated EC and SMC from pig aorta [7, 31]. Although not shown, the following controls gave no PCR products: no reverse transcriptase and no cDNA template. Identities of PMCA1b and PMCA4b bands were also confirmed by the sequencing of gel-purified PCR products. Specificity of the PMCA4a was confirmed by RT-PCR with PMCA4a specific primers (Table 1).

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