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Abstract-Subplasmalemmal Ca 2ϩ , dynamically equilibrated with extracellular Ca 2ϩ , affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca 2ϩ wave propagating beneath the plasma membrane in response to acute elevation of extracellular [Ca 2ϩ ], by targeting a Ca 2ϩ sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca 2ϩ waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLC␦ from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PH␦ translocation, but also the peak amplitude of the subcortical Ca 2ϩ waves; this finding suggests the involvement of local IP 3 production through PLC-mediated PIP 2 hydrolysis in the initiation of these waves. Changes in NO production as well as PKC␤-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA 2 to perinuclear endomembranes, were associated with the subplasmalemmal Ca 2ϩ changes.

Circulating NEFAs (non-esterified fatty acids) from adipose tissue lipolysis lead to endothelial dysfunction and insulin resistance in patients with the metabolic syndrome or Type 2 diabetes mellitus. The aim of the present study was to test the hypothesis that DHP (dihydropyridine) CCBs (calcium channel blockers) prevent NEFA-induced endothelial and haemorheological dysfunction independently of their antihypertensive properties. Using a double-blind cross-over study design, nifedipine, amlodipine, diltiazem or placebo were administered to eight healthy subjects for 2 days before each study day. On the study days, the following were assessed before and after the infusion of lipid and heparin to raise serum NEFAs: endothelial function, by measuring FBF (forearm blood flow) responses to ACh (acetylcholine); leucocyte activation, by ex vivo measurement of plasma MPO (myeloperoxidase) levels, adherent leucocyte numbers and whole blood transit time through microchannels; and oxidative stress, by determining plasma levels of d-ROMs (derivatives of reactive oxygen metabolites). Effects of the CCBs on NF-κB (nuclear factor κB) p65 phospholylation stimulated by NEFAs were assessed in cultured monocytic cells in vitro. Elevated NEFAs reduced the responses to ACh and significantly increased whole blood transit time, adherent leucocyte numbers and d-ROMs. Nifedipine and amlodipine, but not diltiazem, prevented NEFA-induced endothelial dysfunction, leucocyte activation and enhancement of oxidative stress without affecting BP (blood pressure), whereas all these drugs prevented NEFA-induced p65 activation in vitro. These results suggest that DHP CCBs, independent of their antihypertensive properties in humans, prevent NEFA-induced endothelial and haemorheological dysfunction through inhibition of NEFA-induced leucocyte activation, although the sensitivity to drugs of leucocyte Ca2+ channels may differ among cells.