Biomedical subjects

Kanwaldeep K Mallhi

Publications and source records attributed to Kanwaldeep K Mallhi.

4 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Ca2+-mediated ascorbate release from coronary artery endothelial cells.

1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.

Adenosine Triphosphate↗

PubMed2005

A novel plasma membrane Ca(2+)-pump inhibitor: caloxin 1A1.

The plasma membrane Ca(2+)-Mg(2+)-ATPase is a Ca(2+)-pump that expels Ca2+ from cells. Here we report caloxin 1A1-a novel peptide inhibitor (Ki=100 microM) of plasma membrane Ca(2+)-pump-obtained by screening a cysteine bridge-constrained random peptide library for binding to the first extracellular domain of plasma membrane Ca(2+)-pump. Dithiothreitol removed the inhibition indicating that the constraint imposed by the cysteine bridge is required for the inhibition. Caloxin 1A1 also inhibited the fast twitch sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca(2+)-Mg(2+)-ATPase activity of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, but not that of plasma membrane Ca(2+)-pump. Caloxin 1A1 stabilised Ca(2+)-dependent formation of the acid stable 140-kDa acylphosphate which is a partial reaction of this enzyme. Thus caloxin 1A1 inhibits the plasma membrane Ca(2+)-pump by perturbing the first extracellular domain indicating that the transmembrane domains 1 and 2 play a role in its reaction cycle. This finding is consistent with rearrangements that occur in transmembrane helices 1 and 2 during reaction cycle of sarcoplasmic reticulum Ca(2+)-pump. Caloxin 1A1 caused an increase in cytosolic Ca2+ concentration in endothelial cells.

Amino Acid Sequence↗

PubMed2005

Role of third extracellular domain of plasma membrane Ca2+-Mg2+-ATPase based on the novel inhibitor caloxin 3A1.

The plasma membrane Ca2+ pump (PMCA) is a Ca2+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ ([Ca2+]i). It contains five putative extracellular domains (PEDs). Earlier we had reported that binding to PED2 leads to PMCA inhibition. Mutagenesis of residues in transmembrane domain 6 leads to loss of PMCA activity. PED3 connects transmembrane domains 5 and 6. PED3 is only five amino acid residues long. By screening a phage display library, we obtained a peptide sequence that binds this target. After examining a number of peptides related to this original sequence, we selected one that inhibits the PMCA pump (caloxin 3A1). Caloxin 3A1 inhibits PMCA but not the sarcoplasmic reticulum Ca2+-pump. Caloxin 3A1 did not inhibit formation of the 140 kDa acylphosphate intermediate from ATP or its degradation. Thus, PEDs play a role in the reaction cycle of PMCA even though sites for binding to the substrates Ca2+ and Mg-ATP2-, and the activator calmodulin are all in the cytosolic domains of PMCA. In endothelial cells exposed to low concentration of a Ca2+-ionophore, caloxin 3A1 caused a further increase in [Ca2+]i proving its ability to inhibit PMCA pump extracellularly. Thus, even though PED3 is the shortest PED, it plays key role in the PMCA function.

Animals↗