Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions

Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca²⁺, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca²⁺ concentration, [Ca²⁺](i), is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP₃), a second messenger that can evoke Ca²⁺ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca²⁺ and InsP₃ in freshly isolated pairs or small clusters of rat hepatocytes. The Ca²⁺ indicator fura-2 was used to monitor transjunctional diffusion of Ca²⁺ directly or to detect passage of InsP₃ by localized Ca²⁺ release. Fura-2 injected as the free acid passed between cells. Injection of InsP₃ or CaCl₂ immediately increased [Ca²⁺](i) in the injected cell (peak values < 1 μM), and [Ca²⁺](i) increased rapidly in contacting cells (within seconds). The initial rise in [Ca²⁺](i) induced by InsP₃ was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca²⁺. In the coupled cells the regions of greatest increase were not necessarily near the contact zone. In contrast, the rise induced in [Ca²⁺](i) by CaCl₂ injection when cells were bathed in normal Ca²⁺ was always more diffuse than with InsP₃ injection, and in cells coupled to a cell injected with CaCl₂ the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP₃ (or an active metabolite, but not Ca²⁺) diffused between cells to cause localized release of Ca²⁺ from intracellular stores. Ca²⁺ injection induced a rise in [Ca²⁺](i) in coupled cells even when cells were maintained in Ca²⁺-free saline, suggesting that changes in [Ca²⁺](i) seen in adjacent cells were due to transjunctional diffusion from the injected cells and not to uptake from the extracellular solution. However, in Ca²⁺-free saline, [Ca²⁺](i) distribution was nonuniform, indicating that Ca²⁺-releasing mechanisms contribute to the observed changes. No increase in [Ca²⁺](i) was seen in adjacent cells when Ca²⁺ was injecred after treatment with the uncoupling agent octanol (500 μM), which itself did not change [Ca²⁺](i). These data provide evidence that the second messengers Ca²⁺ and InsP₃ can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.