VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR₁₋₂) in endothelial cells with a synthetic ligand that binds specifically to VEGFR₁₋₂. The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR₁₋₂ activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR₁₋₂ activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR₁₋₂ inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Endothelial cells (ECs) are regulated not only by circulating hormones, but also by mechanical stresses, such as shear force. Ion channels in ECs can signal rapid changes of shear forces and are involved in controling EC permeability, proliferation, and angiogenesis. In this study, we employed patch clamping and molecular biology approaches to clarify whether the epithelial sodium channel (ENaC) is functionally expressed in ECs. The α‐subunit of the ENaC was expressed in cultured human ECs and in intact ECs from a variety of rat arteries. In either inside‐ or outside‐out current recordings, inward currents with a conductance of 4.83 pS were detected in cultured human ECs, where these were sensitive to micromolar amiloride. The right shift of the I‐V curve in the condition of low cytoplasmic Na⁺implicated that these currents were carried by Na⁺. The currents were mediated by ENaC channels, as confirmed by ENaC knockdown experiments. However, the activity of ENaC was nearly absent in intact ECs, because its activity was greatly inhibited by cellular molecules, partly due to 11,12‐epoxyeicosatrienoic acid. In the outside‐out configuration, laminar flow directly enhanced ENaC opening probability, suggesting a potential role for ENaC in mediating shear force signaling events.

Osteoblasts and osteoclasts express functionalN-methyl-d-aspartate (NMDA) receptors, which participate in regulation of bone matrix. In rat femoral osteoblasts held in whole cell clamp there is a robust NMDA current but little if any response to l-glutamate. We have investigated expression of metabotropic glutamate receptors (mGluRs) in these cells. By reverse transcription polymerase chain reaction, we have detected expression of mGluR1b (but not mGluR1a, 2, 3, 4, 5, or 6). Blockade of mGluRs with (±)-α-methyl-carboxyphenyl-glycine resulted in an enlarged l-glutamate-induced current that resembled the response to NMDA. Conversely, prior stimulation of mGluRs withtrans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD; mGluR agonist) reduced the NMDA-induced current by 77%. Monitoring of [Ca²⁺]_i showed that NMDA induced a sustained elevation of [Ca²⁺]_i, which was dependent upon [Ca²⁺]_o. Treatment with 1S,3R-ACPD generated an initial transient that was independent of [Ca²⁺]_o, followed by a sustained, [Ca²⁺]_o-dependent phase, a response consistent with phospholipase C-mediated mobilization of stored Ca²⁺. Investigations of the interaction between the two receptors confirmed inhibitory modulation of the NMDA receptor-induced rise in [Ca²⁺]_i by mGluRs. Parathyroid hormone, which also activates phospholipase C in osteoblasts, had a similar inhibitory effect on the NMDA receptor-induced [Ca²⁺]_i response. Elevation of [Ca²⁺]_i mediated by mGluR activation was reduced by subsequent stimulation of NMDA receptors. This is the first description of mGluRs in bone and shows that complex glutamatergic signaling can occur in this tissue.