Aortic smooth muscle and endothelial plasma membrane Ca²⁺ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Abstract

Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension

“…In contrast, others have shown that mRNA for PMCA1 is more prevalent than that for PMCA4 in rat cultured aortic VSMCs, with PMCA4 transcripts suggested to be at only a quarter of the level of those for PMCA1 (Sasamura et al, 2002). In porcine aortic endothelial cells PMCA1 RNA has been shown to be detectable at a higher level than PMCA4 transcripts (Pande et al, 2006;Szewczyk et al, 2007). Thus, the vascular expression of PMCA1 and 4 may be species specific, with further work being required to clearly determine the relative expression of PMCA1 and PMCA4 in VSMCs and endothelial cells, particularly in resistance arteries.…”

“…However, Pande and colleagues have suggested that PMCA4 is the dominant form in pig aortic VSMCs (Pande et al, 2006). In contrast, others have shown that mRNA for PMCA1 is more prevalent than that for PMCA4 in rat cultured aortic VSMCs, with PMCA4 transcripts suggested to be at only a quarter of the level of those for PMCA1 (Sasamura et al, 2002).…”

“…PMCA1 RNA has been detected in pig aortic smooth muscle and endothelial cells (Pande et al, 2006), whilst mRNA for PMCA1 has also been detected in rat cultured aortic VSMCs (Sasamura et al, 2002). At the protein level, PMCA1 is present in VSMCs from mouse aorta (Kobayashi et al, 2012), as well as in endothelial cells from the pig coronary artery (Szewczyk et al, 2007).…”

“…A number of caloxin compounds have been designed, with screens against PMCA4 identifying caloxin 1b1 as an inhibitor of PMCA4 (K i = 46 ± 5 μM compared K i = 105 ± 11 μM for PMCA1) (Pande et al, 2006). By binding to the first extracellular domain of PMCA4 it is proposed that caloxin 1b1 inhibits Ca 2+ flux non-competitively by blocking/altering the required conformational change in the protein which occurs with the binding of ATP (Holmes et al, 2003;Pande et al, 2006).…”

Measurement of Plasma Membrane Calcium–Calmodulin-Dependent ATPase (PMCA) Activity

“…The use of human erythrocyte ghosts was common in the preparation of purified PMCA for this purpose (9). However, this will yield mainly PMCA4 as this is the main isoform expressed in human red blood cells.…”