Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1.
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.
Pande
Jyoti
J
Department of Medicine, HSC 4N41, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada.
Mallhi
Kanwaldeep K
KK
Sawh
Ahilya
A
Szewczyk
Magdalena M
MM
Simpson
Fiona
F
Grover
Ashok K
AK
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000008:administration & dosage*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011921:Rats, Inbred WKY; D013329:Structure-Activity Relationship
D010455:Peptides; D020033:Protein Isoforms; C510327:caloxin 1b1; D000252:Calcium-Transporting ATPases