Agonist-stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells

Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]( i)) was measured by digital imaging microscopy. Agonists ATP (100 mu M), th rombin (10 units/ml), and histamine (25 mu M) induced a transient [Ca2+](i) increase. Histamine (100 mu M) induced a biphasic [Ca2+](i) increase with an initial [Ca2+](i) peak followed by a [Ca2+](i) plateau. The [Ca2+](i) pl ateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to th e [Ca2+](i) plateau. However, this [Ca2+](i) plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization w ith 80K(+) or application of the VGC agonist BAY K 8644 did not alter the r esting [Ca2+](i); but 80K+ reduced the histamine (100 mu M) induced [Ca2+]( i), plateau. These results show that HCMEC are devoid of functional VGC. Th us the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+](i). Blockade of sarcoplasmic/endoplasmic r eticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+](i). This effect was de pendent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide sy nthase (eNOS), which stimulates cyclic GMP production. The final result tha t the eNOS inhibitor L-NAME enhanced the histamine (100 mu M) induced [Ca2](i), plateau suggests a negative feedback loop (via cGMP) of endothelial N O on its own synthesis in the regulation of endothelial [Ca2+] signal. (C) 1999 Academic Press.