Inside-Out IP3-Mediated G Protein-Coupled Receptor Activation Drives Intercellular Ca2+ Signaling in the Vascular Endothelium

The endothelium's control of nearly all vascular function relies on rapid intercellular communication to coordinate cellular activity across scale. A key form of intercellular communication arises from the regenerative propagation of IP₃-evoked Ca²⁺ signals from cell to cell, which regulate vessel tone, modulate vascular permeability, and determine immune responses. Despite their importance, the mechanisms by which regenerative propagation of IP₃-evoked Ca²⁺ signals occurs are poorly understood. Here, in intact resistance arteries, precision photolysis of IP₃ combined with high-resolution mesoscale imaging, targeted drug application, and advanced analytical techniques was used to determine the mechanisms underlying regenerative propagation of IP₃-evoked Ca²⁺ signals in the endothelium. Elevated IP₃ in the initiating cell triggers a noncanonical inside-out signaling mechanism that leads to transcellular activation of a G_αq/11-coupled receptor in a neighboring (receiving) cell. This, in turn, initiates canonical outside-in signaling via PLC, leading to the hydrolysis of PIP₂ and production of IP₃. This process creates a regenerative, IP₃-dependent signaling cascade operating between adjacent cells. Notably, neither Ca²⁺ nor IP₃ diffusion through gap junctions plays a significant role in intercellular communication. Our findings uncover a previously unrecognized mechanism of endothelial communication, in which noncanonical IP₃-driven transcellular activation of G protein-coupled receptors sustains a regenerative signaling loop, highlighting a novel framework for intercellular coordination in the vascular endothelium.