Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation.

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 µM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

Arachidonic Acid Evokes an Increase in Intracellular Ca2+ Concentration and Nitric Oxide Production in Endothelial Cells from Human Brain Microcirculation.

It has long been known that the conditionally essential polyunsaturated arachidonic acid (AA) regulates cerebral blood flow (CBF) through its metabolites prostaglandin E2 and epoxyeicosatrienoic acid, which act on vascular smooth muscle cells and pericytes to vasorelax cerebral microvessels. However, AA may also elicit endothelial nitric oxide (NO) release through an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we adopted Ca2+ and NO imaging, combined with immunoblotting, to assess whether AA induces intracellular Ca2+ signals and NO release in the human brain microvascular endothelial cell line hCMEC/D3. AA caused a dose-dependent increase in [Ca2+]i that was mimicked by the not-metabolizable analogue, eicosatetraynoic acid. The Ca2+ response to AA was patterned by endoplasmic reticulum Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptors, lysosomal Ca2+ mobilization through two-pore channels 1 and 2 (TPC1-2), and extracellular Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4). In addition, AA-evoked Ca2+ signals resulted in robust NO release, but this signal was considerably delayed as compared to the accompanying Ca2+ wave and was essentially mediated by TPC1-2 and TRPV4. Overall, these data provide the first evidence that AA elicits Ca2+-dependent NO release from a human cerebrovascular endothelial cell line, but they seemingly rule out the possibility that this NO signal could acutely modulate neurovascular coupling.