OBJECTIVETo study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels.

METHODBALB/c mice were used for this experiment. Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated, and were divided into a negative control group, a Con A group, and 0.04, 0.4, 4, and 20 mmol x L(-1) betaine groups. MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes; flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes; and laser confocal scanning microscopy was used to observe the changes in the intracellular [Ca2+]i of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied.

RESULTBetaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol x L(-1). It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0.04, 0.4, 4, and 20 mmol x L(-1), with the effect being most marked for the 4 mmol x L(-1) group 24 h after its application. It was found to facilitate the entry of mouse spleen lymphocytes from the G0/G1 to the S phase 4, 6, 18, and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol x L(-1), with the effect being most marked at 18 h after its application. Intracellular [Ca2+]i in mouse spleen lymphocytes increased significantly (P < 0.01) 6, 12, 18 h after 4 mmol x L(-1) betaine had acted on the lymphocytes, with the effect being most marked at 6 h. The calcium channel blockers nifidipine, diltiazem, mibefradil, and genistein had no effect on the increase of the intracellular [Ca2+]i in mouse spleen lymphocytes due to the application of betaine, while verapamil, mycifradin, heparin, and procaine could block such increase.

CONCLUSIONBetaine facilitates the entry of mouse spleen lymphocytes from the G0/G1 into the S phase by raising the intracellular [Ca2+]i in these cells, thus promoting their proliferation. Intracellular [Ca2+]i increases mainly in two ways: (1) By affecting the alpha1 subunit of the L-type voltage-gated calcium channel with mediation by G proteins and thus leading to an efflux of intracellular calcium: (2) By affecting the IP3R and RyR calcium channels of the intracellular calcium stores and thus leading to the release of intracellular calcium.