Anandamide initiates Ca(2+) signaling via CB(2) receptor linked to phospholipase C in calf pulmonary endothelial cells

1. The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. 2. The present study was designed to explore in detail the effect of this endocannabinoid on Ca(2+) signaling in single cells of a calf pulmonary endothelial cell line. 3. Anandamide initiated a transient Ca(2+) elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. 4. The phospholipase C inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca(2+) signaling in response to anandamide. 5. Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca(2+) depletion of the ER accompanied by the activation of capacitative Ca(2+) entry (CCE) and a transient elevation of mitochondrial Ca(2+). 6. These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca(2+) release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca(2+) elevation was accompanied by a transient Ca(2+) increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells

Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells

The involvement of ryanodine-sensitive Ca2+ release (RsCR) in bradykinin (Bk)-induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells.Using deconvolution microscopy fura-2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP-18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura-2 and FFP-18.Inhibition of Na+−Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk-induced [Ca2+]pn signal in Ca2+-free solution. This effect was abolished when RsCR was prevented with 25 μmmu;mol l−1 ryanodine, while inhibition of RsCR had no effect on Bk-induced increase in [Ca2+]pn without inhibition of Na+−Ca2+ exchange.Initiating RsCR by 200 nmol l−1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+−Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn.Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+-containing solution.Inhibition of RsCR blunted Bk-activated CCE, while inhibition of Na+−Ca2+ exchange during stimulation enhanced CCE.Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged.These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS

Anandamide-induced mobilization of cytosolic Ca(2+) in endothelial cells

1. Experiments were designed to determine whether anandamide affects cytosolic Ca(2+) concentrations in endothelial cells and, if so, whether CB(1) cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca(2+) using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca(2+) in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca(2+) from intracellular stores without triggering capacitative Ca(2+) entry, contrary to histamine or the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca(2+) from intracellular stores that was evoked by histamine. The mobilization of Ca(2+) from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[(3)H]-arginine to L-[(3)H]-citruline. 5. The CB(1) cannabinoid receptor antagonist SR141716A (1 μM) only produced a marginal reduction of the mobilization of Ca(2+) produced by 5 μM anandamide. However, at 5 μM SR141716A elicited the release of Ca(2+) from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca(2+) evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 μM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(−1) pertussis toxin (to uncouple CB(1) cannabinoid receptors from G(i/o) proteins) had no significant effect on the mobilization of cytosolic Ca(2+) evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca(2+) from a caffeine-sensitive intracellular Ca(2+) store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB(1) cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca(2+) in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca(2+) signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone

Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4

TRPV4 is a Ca(2+)- and Mg(2+)-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca(2+)-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5′,6′-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A(2) (PLA(2)) and cytochrome P450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4α-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4α-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA(2)-dependent formation of AA, and its subsequent metabolization to 5′,6′-epoxyeicosatrienoic acid by means of a cytochrome P450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA(2)- and cytochrome P450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain

Functional interaction of endothelial nitric oxide synthase with a voltage-dependent anion channel

Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS) whose function is modulated, in part, by specific protein interactions. By coimmunoprecipitation experiments followed by MS analyses, we identified a human voltage-dependent anion/cation channel or porin as a binding partner of eNOS. The interaction between porin and eNOS was demonstrated by coimmunoprecipitation studies in nontransfected human endothelial cells and Cos-7 cells transiently transfected with eNOS and porin cDNAs. In vitro binding studies with glutathione S-transferase–porin indicated that porin binds directly to eNOS and that this interaction augmented eNOS activity. The calcium ionophore, A23187, and bradykinin, which are known to activate eNOS, markedly increased porin–eNOS interaction, suggesting a potential role of intracellular Ca(2+) in mediating this interaction. Theses results indicate that the interaction between a voltage-dependent membrane channel and eNOS may be important for regulating eNOS activity