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

Abstract

Histamine‐stimulated cytosolic free Ca2+ ([Ca2+]i) oscillations in human umbilical vein endothelial cells (HUVECs) comprise repetitive spikes generated by pulsatile release from stores. We have investigated the roles of the store Ca2+‐ATPases in regulating both the upstroke and downstroke of a Ca2+ spike. The sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) dramatically affected oscillations whereas inhibition of the plasma membrane Ca2+‐ATPase (PMCA) with La3+ had little effect. This and other evidence suggested that the downstroke of a spike is predominantly mediated by SERCA. Artificial [Ca2+]i spiking generated by repetitive pulsatile application of 0.3 μm histamine in Ca2+‐free medium did not cause net loss of Ca2+ from the cell whereas repetitive pulsatile application of 1 and 10 μm histamine did, with the higher concentration being more effective. We conclude that there is an inverse relationship between stimulus intensity and relative SERCA activity. 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 ISP was shortened by low CPA concentrations, (ii) higher concentrations of CPA stimulated an explosive Ca2+ release when applied during the ISP but not when applied in the absence of agonist, and (iii) CPA synchronized the initial Ca2+ response to a low histamine dose (even recruiting silent, histamine‐unresponsive cells). Two aspects of the regenerative upstroke of a spike were differently affected by SERCA inhibition: Ca2+ wave velocity was entirely unaffected by CPA whereas the local rate of rise was increased. The [Ca2+]i at which a Ca2+ spike terminated depended on SERCA since CPA dose dependently enhanced the peak [Ca2+]i. 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+ spike.

Evidence that Ca2+ cycling by the plasma membrane Ca2+-ATPase increases the `excitability' of the extracellular Ca2+-sensing receptor

“…Overall it appears that PMCA inhibition does not have a dramatic effect on oscillations in WT cells. This conclusion is consistent with previous reports of Morgan and Jacob (Morgan and Jacob, 1998) who found that inhibiting the PMCA with La 3+ (which could not be used in the present study because it is an agonist of CaR) had little effect on Ca 2+ oscillations in endothelial cells. Moreover, Green et al, also showed that another PMCA inhibitor, carboxyeosin (which also could not be used here due to its intense fluorescence) did not block Ca 2+ oscillations in rat hepatocytes as measured by the luminescent photoprotein aequorin (Green et al, 1997).…”

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells

“…However, the established routes for gated release of Ca 2+ may contribute to a physiological leak, since IP 3 R antagonists have been found to reduce Ca 2+ leakage from the ER after SERCA inhibition in intact cells [24][25][26]. In analogy to previous observations [60,61] …”

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

“…5). A large significant increase in the L p was measured with SKF, TG, and IM treatment, implying that the reason why there was no increase in L p with IM after CPA treatment was because CPA reduced the degree of Ca 2ϩ filling of the store, as expected (20). Once Ca 2ϩ influx was permitted the L p increased again.…”