Gap junction-mediated intercellular diffusion of Ca²⁺ in cultured human corporal smooth muscle cells

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 ± 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca²⁺ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5- trisphosphate [Ins(1,4,5)P₃] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca²⁺ in response to Ins(1,4,5)P₃ was more variable than for Ca²⁺ injections. These studies demonstrate that Ca²⁺, and perhaps Ins(1,4,5)P₃ as well, can diffuse between smooth muscle cells through gap junction channels.