Differential modulation of the phases of a Ca2+ spike by the store Ca2+-ATPase in human umbilical vein endothelial cells

1. Histamine-stimulated cytosolic free Ca2+ ([Ca2+](i)) oscillations in hum an umbilical win endothelial cells (HUVECs) comprise repetitive spikes gene rated by pulsatile release from stores. We have investigated the roles of t he store Ca2+-ATPases in regulating both the upstroke and downstroke of a C a2+ spike. 2. The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazo nic acid (CPA) dramatically affected oscillations whereas inhibition of the plasma membrane Ca2+-ATPase (PMCA) with La3+ had little effect. This and o ther evidence suggested that the downstroke of a spike is predominantly med iated by SERCA. 3. Artificial [Ca2+](i) spiking generated by repetitive pulsatile applicati on of 0.3 mu M histamine in Ca2+-free medium did not cause net loss of Ca2 from the cell whereas repetitive pulsatile application of 1 and 10 mu M hi stamine did, with the higher concentration being more effective. We conclud e that there is an inverse relationship between stimulus intensity and rela tive SERCA activity. 4. For a Ca2+ transient, the initiation of release was suppressed by SERCA during either the lag phase or the interspike period (ISP) since: (i) the I SP was shortened by low CPA concentrations, (ii) higher concentrations of C PA stimulated an explosive Ca2+ release when applied during the ISP but not when applied in the absence of agonist, and (iii) CPA synchronized the ini tial Ca2+ response to a low histamine dose (even recruiting silent, histami ne-unresponsive cells). 5. Two aspects of the regenerative upstroke of a spike were differently aff ected by SERCA inhibition: Ca2+ wave velocity was entirely unaffected by CP A whereas the local rate of rise was increased. 6. The [Ca2+](i) at which a Ca2+ spike terminated depended on SERCA since C PA dose dependently enhanced the peak [Ca2+](i). 7. We conclude that SERCA plays a powerful and dynamic role in regulating [ Ca2+](i) oscillations in HUVECs. SERCA differentially modulates the phases of Ca2+ release in addition to bringing about the falling phase of a Ca2+ s pike.