Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal (GI) tract and have important functions in the control of GI motility. Loss of ICC is associated with several GI motility disorders; yet, the mechanisms modulating ICC survival and proliferation are not fully understood. Hydrogen sulfide (H(2)S) has been reported to be a gaseous transmitter that regulates cellular proliferation. This study aims to establish whether H(2)S participates in regulation of ICC proliferation. The effect of H(2)S was studied in primary cultures of ICC, prepared from the mouse small intestine. To determine the extent of ICC proliferation, we used immunofluorescent staining to study alterations in the number of cells expressing c-Kit(+) and CD44(+), markers for mature ICC. Phosphorylation of Akt was measured by Western blot analysis. Treatment with low concentrations of NaHS (H(2)S donor, 1-30 microM) showed no apparent toxicity, as judged from cell numbers. Importantly, treatment with NaHS (15 microM) for 24 hours increased the numbers of c-Kit(+)/CD44(+) ICC by 23.3 +/- 1.4% (P < 0.05). Moreover, NaHS increased Akt phosphorylation, which was prevented with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (5 microM). LY294002 also blocked the NaHS-mediated increase in the number of ICC. In addition, H(2)S enhanced the proliferation of mature ICC in the in vitro culture system used here in a concentration-dependent manner. The present study suggests that H(2)S may be a critical factor in maintaining ICC numbers and may have a novel, Akt-dependent role in proliferation of mature ICC.